Functional antibody fragment complementation for a two-components system for redirected killing of unwanted cells

ABSTRACT

A targeted T-cell engaging agent for treating a condition characterized by the presence of unwanted cells includes (a) a targeting moiety that is capable of targeting the unwanted cells; (b) a first T-cell engaging domain capable of T-cell engaging activity when binding a second T-cell engaging domain, wherein the second T-cell engaging domain is not part of the agent; (c) at least one inert binding partner capable of binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed; and (d) at least one cleavage site separating the first T-cell engaging domain and the inert binding partner, wherein the cleavage site is: (i) cleaved by an enzyme expressed by the unwanted cells; (ii) cleaved through a pH-sensitive cleavage reaction inside the unwanted cell; (iii) cleaved by a complement-dependent cleavage reaction; or (iv) cleaved by a protease that is colocalized to the unwanted cell by a targeting moiety that is the same or different from the targeting moiety in the agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of U.S. Provisional Application No. 62/257,552, filed Nov. 19, 2015, and U.S. Provisional Application No. 62/270,907, filed Dec. 22, 2015.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “2017-04-19_01131-0007-00US_SL_ST25.txt” created on Apr. 19, 2017, which is 84,197 bytes in size.

FIELD

This application relates to targeted T-cell engaging agents for treating a condition characterized by the presence of unwanted cells. In particular, it relates to agents that can be used to treat a condition characterized by the presence of unwanted cells, such as cancer or other disease-causing cells.

BACKGROUND

Cancer and other diseases caused by the presence of unwanted cells create significant loss of life, suffering, and economic impact. Immunotherapeutic strategies for targeting cancer have been an active area of translational clinical research.

A variety of other approaches have been explored for immunotherapy, but many of these prior approaches lack sufficient specificity to particular unwanted cells. For example, demibodies have been designed each having an scFv portion binding to different antigens on a target cell, an Fc domain allowing pairing to a complementary demibody, and a binding partner capable of forming an association to another binding partner on a complementary demibody. WO 2007/062466. These demibodies, however, are not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens. See also WO 2013/104804, which provides a first polypeptide with a targeting moiety binding to a first antigen and a first fragment of a functional domain, along with a second polypeptide with a targeting moiety binding to a second antigen and a second fragment of a functional domain that is complementary to the first fragment of the functional domain. Likewise, this approach is not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens.

While some positive test data has been shown with prior approaches, clinically-effective therapeutic strategies must be able to elicit a strong immune response in an individual suffering from a disease such as cancer. Additionally, effective therapies should be very specific and not cause unwanted side effects to other cell types in the body. Therefore, additional developments in this field of re-directed immunotherapy are required.

SUMMARY

In accordance with the description, the inventors describe a targeted T-cell engaging agent for treating a condition characterized by the presence of unwanted cells. This agent includes (a) a targeting moiety that is capable of targeting the unwanted cells; (b) a first T-cell engaging domain capable of activity when binding a second T-cell engaging domain, wherein the second T-cell engaging domain is not part of the agent; (c) at least one inert binding partner capable of binding the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed; and (d) at least one cleavage site separating the first T-cell engaging domain and the inert binding partner.

In one embodiment, a two-component system for treating a condition characterized by the presence of unwanted cells is encompassed comprising a first component comprising a targeted T-cell engaging agent comprising:

-   -   a. a first component comprising a targeted T-cell engaging agent         comprising         -   i. a first targeting moiety that is capable of targeting the             unwanted cells;         -   ii. a first T-cell engaging domain capable of T-cell             engaging activity when binding a second T-cell engaging             domain, wherein the second T-cell engaging domain is not             part of the first component;         -   iii. a first inert binding partner for the first T-cell             engaging domain binding to the first T-cell engaging domain             such that the first T-cell engaging domain does not bind to             the second T-cell engaging domain unless the inert binding             partner is removed; and         -   iv. a cleavage site separating the first T-cell engaging             domain and the first inert binding partner, wherein the             cleavage site is:             -   (1) cleaved by an enzyme expressed by the unwanted                 cells;             -   (2) cleaved through a pH-sensitive cleavage reaction                 inside the unwanted cell;             -   (3) cleaved by a complement-dependent cleavage reaction;                 or             -   (4) cleaved by a protease that is colocalized to the                 unwanted cell by a targeting moiety that is the same or                 different from the targeting moiety in the agent,     -   b. a second component comprising a second T-cell engaging domain         capable of T-cell engaging activity when binding the first         T-cell engaging domain, wherein the first and second T-cell         engaging domains are capable of binding when neither is bound to         an inert binding partner.

In another embodiment, the second component of the two-component system further comprises a second targeting moiety that is capable of targeting the unwanted cells.

In another embodiment, the second component of the two-component system further comprises a second inert binding partner for the second T-cell engaging domain binding to the second T-cell engaging domain such that the second T cell engaging domain does not bind to the first T-cell engaging domain unless the inert binding partner is removed and

-   -   a. a cleavage site separating the second T-cell engaging domain         and the second inert binding partner, wherein the cleavage site         is:         -   i. cleaved by an enzyme expressed by the unwanted cells;         -   ii. cleaved through a pH-sensitive cleavage reaction inside             the unwanted cell;         -   iii. cleaved by a complement-dependent cleavage reaction; or         -   iv. cleaved by a protease that is colocalized to the             unwanted cell by a targeting moiety that is the same or             different from the targeting moiety in the agent,             wherein cleavage of the cleavage site causes loss of the             inert binding partner and complementation with the first             T-cell engaging domain of the two-component system.

In some embodiments, the first and second targeting moieties of the two-component system are the same.

In some embodiments, the first and second targeting moieties of the two-component system are different

In some embodiments, the first and second cleavage sites are the same.

In some embodiments, the first and second cleavage sites are different.

In some embodiments, at least one cleavage site is a protease cleavage site. In some embodiments, the at least one cleavage site is capable of being cleaved outside the unwanted cells.

In some embodiments of the two-component system, at least one enzyme expressed by the unwanted cells is a protease.

In some embodiments of the two-component system, at least one inert binding partner specifically binds the T-cell engaging domain.

In some embodiments of the two-component system, at least one inert binding partner is a VH or VL domain.

In some embodiments of the two-component system, the T-cell engaging domain is a VH domain, the inert binding partner is a VL domain and when the T-cell engaging domain is a VL domain, the inert binding partner is a VH domain.

In some embodiments of the two-component system, at least one targeting moiety is an antibody or functional fragment thereof. In some embodiments of the two-component system, the at least one inert binding partner is capable of dissociation once at least one cleavage site has been cleaved and after dissociation the two T-cell engaging domains are capable of binding to each other and exhibiting T-cell engaging activity.

In some embodiments of the two-component system, a set of nucleic acid molecules encodes the first and second component of the two-component system. In some embodiments of the two-component system, a nucleic acid molecule encodes the component for use in a two-component system.

In some embodiments of the two-component system, one T-cell engaging domain is a VH domain and the other T-cell engaging domain is a VL domain.

In another embodiment, a component for use in a two-component system for treating a condition characterized by the presence of unwanted cells comprising a first targeted T-cell engaging agent comprises:

-   -   a. a targeting moiety that is capable of targeting the unwanted         cells;     -   b. a first T-cell engaging domain capable of T-cell engaging         activity when binding a second T-cell engaging domain, wherein         the second T-cell engaging domain is not part of the first         targeted T-cell engaging agent;     -   c. an inert binding partner for the first T-cell engaging domain         binding to the first T-cell engaging domain such that the first         T-cell engaging domain does not bind to the second T-cell         engaging domain unless the inert binding partner is removed; and     -   d. a cleavage site separating the first T-cell engaging domain         and the inert binding partner, wherein the cleavage site is:         -   i. cleaved by an enzyme expressed by the unwanted cells;         -   ii. cleaved through a pH-sensitive cleavage reaction inside             the unwanted cell;         -   iii. cleaved by a complement-dependent cleavage reaction; or             cleaved by a protease that is colocalized to the unwanted             cell by a targeting moiety that is the same or different             from the targeting moiety in the agent, wherein cleavage of             the cleavage site causes loss of the inert binding partner             and allows for complementation with the second T-cell             engaging domain that is not part of the agent.

In some embodiments, a method of treating a disease in a patient characterized by the presence of unwanted cells is encompassed that comprises administering the two-component system to the patient. In some embodiments, a method of targeting an immune response of a patient to unwanted cells is encompassed that comprises administering the two-component system. In some embodiments, these unwanted cells are cancer cells. In some embodiments, the cancer is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.

Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of a first component of a two-component system, where the first component is a targeted T-cell engaging agent in an inactive state with an inert binding partner.

FIG. 2 shows the process by which the cleavable linker is cleaved and the inert binding partner is released to create an active entity.

FIG. 3 illustrates the creation of an active targeted, T-cell engaging agent after the inert binding partner is released from a pair of complementary components in a two-component system.

FIGS. 4A-C illustrate the cleavage of the stepwise process of the pair of complementary components in a two-component system binding to the target cell (A), cleavage of linker attaching the inert binding partners (A and B), and binding to create an active moiety capable of T-cell

FIGS. 5A-B provide evaluation of constructs by SDS PAGE and Coomassie blue staining.

FIG. 6 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with various individual constructs and combinations, with 6245 serving as a positive control and the combination of 6248 and 6249 showing beneficial results.

FIG. 7 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with various individual constructs and combinations, with 6245 as a positive control and the combination of 6248 and 6249 showing beneficial results.

FIG. 8 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with different concentrations of constructs, with 6245 as a positive control and the combination of 6248 and 6249 showing beneficial results.

FIGS. 9A-B shows IFNγ expression as a proxy for T cell response when cancer cells were treated with controls or different concentrations of constructs, with 6245 as a positive control and the combination of 6248 and 6249 showing beneficial results. PHA also served as a positive control for nonspecific T-cell activation.

FIG. 10 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with controls or different concentrations of constructs, with very low levels with constructs having only a VH or VL for the anti-CDE3 scFv, but positive control bispecific constructs (both 9332 and 9333) showed higher levels of activity.

FIG. 11 provides a stoichiometric assessment of complementary constructs of a two-component system.

FIG. 12 shows IFNγ expression as a proxy for T cell response when MCF-7 cancer cells were treated with controls or different concentrations of constructs.

FIG. 13 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with controls or different concentrations of constructs targeting EpCAM.

FIG. 14 shows IFNγ expression as a proxy for T cell response when cancer cells were treated with controls or different concentrations of constructs targeting either biparatopic EGFR epitopes or a combination of EpCAM and EGFR targeting.

FIG. 15 shows the impact of protease inhibitors on constructs either containing protease cleavage sites or not containing protease cleavage sites.

FIG. 16 shows that different types of targeting moieties may be used, by successfully pairing a construct having a VHH targeting moiety with a construct having an scFv moiety.

FIG. 17 shows a sequence schematic for constructs 6248 and 6249 with the various linkers boxed and the protease cleavage site in bold and underline. The His tag is also in bold.

DESCRIPTION OF THE SEQUENCES

Tables 1A and 1B provide a listing of certain sequences referenced herein.

TABLE 1A Description of the Sequences and SEQ ID NOs Description Sequence # ADAM28 cleavage site KPAKFFRL 1 ADAM28 cleavage site DPAKFFRL 2 ADAM28 cleavage site KPMKFFRL 3 ADAM28 cleavage site LPAKFFRL 4 ADAM28 cleavage site LPMKFFRL 5 ADAM28 cleavage site KPAMFFRL 6 ADAM28 cleavage site YPAKFFRL 7 ADAM28 cleavage site KWAKFFRL 8 ADAM28 cleavage site DPMKFFRL 9 ADAM28 cleavage site DPAMFFRL 10 ADAM28 cleavage site DPMMFFRL 11 ADAM28 cleavage site KMAMFFRL 12 ADAM28 cleavage site KMAMFFIM 13 ADAM28 cleavage site KPAMFFIM 14 ADAM28 cleavage site LPAMFFRL 15 ADAM28 cleavage site LPMMFFRL 16 ADAM28 cleavage site LMAMFFRL 17 ADAM28 cleavage site LMAMFFIM 18 ADAM28 cleavage site LPAMFFIM 19 ADAM28 cleavage site LPAMFFYM 20 ADAM28 cleavage site KPMMFFRL 21 ADAM28 cleavage site KPAKFFYM 22 ADAM28 cleavage site KPAKFFIM 23 ADAM28 cleavage site IPMKFFRL 24 ADAM28 cleavage site IPAMFFRL 25 ADAM28 cleavage site IPMMFFRL 26 ADAM28 cleavage site IMAMFFRL 27 ADAM28 cleavage site IMAMFFIM 28 ADAM28 cleavage site IPAMFFIM 29 ADAM28 cleavage site IPAMFFYM 30 cathepsin B cleavage site FR 31 cathepsin B cleavage site FK 32 cathepsin B cleavage site VA 33 cathepsin B cleavage site VR 34 cathepsin B cleavage site V{Cit} 35 cathepsin B cleavage site HLVEALYL 36 cathepsin B cleavage site SLLKSRMVPNFN 37 cathepsin B cleavage site SLLIARRMPNFN 38 cathepsin B cleavage site KKFA 39 cathepsin B cleavage site AFKK 40 cathepsin B cleavage site QQQ 41 cathepsin D cleavage site PRSFFRLGK 42 cathepsin D cleavage site SGVVIATVIVIT 43 cathepsin K cleavage site GGP 44 MMP1 cleavage site SLGPQGIWGQFN 45 MMP2 cleavage site AIPVSLR 46 MMP2 cleavage site SLPLGLWAPNFN 47 MMP2 cleavage site HPVGLLAR 48 MMP2 cleavage site GPLGVRGK 49 MMP2 cleavage site GPLGLWAQ 50 MMP3 cleavage site STAVIVSA 51 MMP7 cleavage site GPLGLARK 52 MMP7 cleavage site RPLALWRS 53 MMP7 cleavage site SLRPLALWRSFN 54 MMP2/9 cleavage site GILGVP 55 MMP2/9 cleavage site GPLGIAGQ 56 MMP9 cleavage site AVRWLLTA 57 MMP9 cleavage site PLGLYAL 58 MMP9 cleavage site GPQGIAGQR 59 MMP9 cleavage site KPVSLSYR 60 MMP11 cleavage site AAATSIN 61 MMP11 cleavage site AAGAMFLE 62 MMP13 cleavage site GPQGLAGQRGIV 63 MMP14 cleavage site PRHLR 64 MMP14 cleavage site PQGLLGAPGILG 65 MMP14 cleavage site PRSAKELR 66 PSA / KLK3 HSSKLQ 67 PSA / KLK3 SSKLQ 68 KLK4 RQQR 69 TMPRSS2 GGR 70 Legumain AAN 71 ST14 (Matriptase) QAR 72 C1s cleavage site YLGRSYKV 73 C1s cleavage site MQLGRX 74 MASP2 cleavage site SLGRKIQI 75 C2a and Bb cleavage site GLARSNLDE 76 uPa cleavage site TYSRSRYL 77 uPa cleavage site KKSPGRVVGGSV 78 uPa cleavage site NSGRAVTY 79 uPa cleavage site AFK 80 tissue-type plasminogen activator GGSGQRGRKALE 81 (tPA) ADAM10 PRYEAYKMGK 82 ADAM12 LAQAF 83 ADAM17 EHADLLAVVAK 84 flexible amino acid linker (may be GGGGS 85 presented in repeating fashion) flexible amino acid linker (may be GGGS 86 presented in repeating fashion) flexible amino acid linker (may be GS 87 presented in repeating fashion) flexible amino acid linker (may be GSGGS 88 presented in repeating fashion) flexible amino acid linker (may be GGSG 89 presented in repeating fashion) flexible amino acid linker (may be GGSGG 90 presented in repeating fashion) flexible amino acid linker (may be GSGSG 91 presented in repeating fashion) flexible amino acid linker (may be GSGGG 92 presented in repeating fashion) flexible amino acid linker (may be GGGSG 93 presented in repeating fashion) flexible amino acid linker (may be GSSSG 94 presented in repeating fashion) Anti-EGFR aptamer (tight binder UGCCGCUAUAAUGCACGGAUUUAAUC 95 with K_(d) = 2.4 nM) GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGGCGCUAAAUAGCACGGAAAUAAUC 96 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCUAGUAUAUCGCACGGAUUUAAUC 97 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGCCAUAUCACACGGAUUUAAUC 98 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UUCCGCUGUAUAACACGGACUUAAUC 99 GCCGUAGUAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGUCGCUCUAUUGCACGGAUUUAAUC 100 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCUGCUUUAUCCCACAUAUUUUUUC 101 CCCUCAUAACAAUAUUUCUCCCCCC Anti-EGFR aptamer UGCNGCUAUAUCGCNCGUAUUUAAUC 102 GCCGUAGAAAAGCAUGUCNANGCCG Anti-EGFR aptamer UGCAAAGAAAACGCACGUAUUUAAUC 103 GCCGUAGUAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAUCACUAUCGAACCUAUUUAAUC 104 CACCAAAAUAAUUGCAAGUCCAUACU C Anti-EGFR aptamer UGCCNNAAUAACACACNUAUAUAAUC 105 GCCGUACAAAAUCAUGUCAAANCCG Anti-EGFR aptamer UGCAGCUGUAUUGCACGUAUUUAAUC 106 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UUCCGAUAAUCCCGCGUACUAAAUCA 107 CCAUAGUCAACAAUUUCCAACCUC Anti-EGFR aptamer UCCACUAUAUCACACGUAUUUAAUCG 108 CCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UCCCUCAACCUCGCUACUAUUUAAUC 109 GCCGUAGAAAAGCAUGUCAAAGCCU Anti-EGFR aptamer UGCCGCUAUAUCACACGAAUUUAAUC 110 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer AGCCCCUAGAACACACGGAUUUAAUC 111 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCAAUAUAUAACACGGAAUUAAUC 112 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGCUAUAGCGCACGGAUUUAAUC 113 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAGAUAUAUGUCACUCAUUAAUCC 114 CCGUAUAAAAACAUAACUAAGCUC Anti-EGFR aptamer UGUAGCUGUAUUGCACACAUUAAAUC 115 GCCGUAGUAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UACCAAUAUAUCGCCACACAUAAUCG 116 CCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGCUAUGCCCACGGAAUUUAAUC 117 GCCGUAGAAAAACAUGUCAAAGUCG Anti-EGFR aptamer UGCCGCUAUUUAGCACGGAUUAAAUC 118 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGCUAUUUAGCACGGAUUAAAUC 119 GCCGUAGAAAAGCAUGUCNAAGCCG Anti-EGFR aptamer UGUAGUAAUAUGACACGGAUUUAAUC 120 GCCGUAGAAAAGCANGUCAAAGCCU Anti-EGFR aptamer UGUCGCCAUUACGCACGGAUUUAAUC 121 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCCCCAAACUACACAAAUUUAAUC 122 GCCGUAUAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCACUAUCUCACACGUACUAAUCGC 123 CGUAUAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGUCGCAAUAAUACACUAAUUUAAUC 124 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAACAAUAUAGCACGUAUUUAAUC 125 GCCGUAGUAAAGCAUGUCAAAGG Anti-EGFR aptamer CUACCACAAAUCCCACAUAUUUAAUC 126 UCCCAAUCAAAUCUUGUCCAUUCCC Anti-EGFR aptamer UGCCCUAAACUCACACGGAUAUAAUC 127 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UUGUCGUAUGUCACACGUAUUAAAUC 128 GCCGUAUAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UUCCGCUAUAACACACGGAGAAAAUC 129 GCCGUAGUAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGAUAUAACGCACGGAUAUAAUC 130 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCAUUAUACAGCACGGAUUUAAUC 131 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UCCAGAAAUAUGCACACAUUUAAUCG 132 CCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UCCGCUAAACAACACGGAUACAAUCG 133 CCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGCACUAUCUCACACGUACUAAUCGC 134 CGUAUAAAAGCAUGUCAAANNNG Anti-EGFR aptamer AUNGCNANNNUACACGUAUUNAAUCG 135 CCGUAGAAAAGCAUGUCANAGCCG Anti-EGFR aptamer UGCUGCUAUAUUGCAAUUUUUUAAAC 136 UAAGUAGAAAACCAUGUACAAGUCG Anti-EGFR aptamer UGUCGCCAUAUUGCACGGAUUUAAUC 137 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGCCGUUAUAACCCACGGAAUUUAAC 138 CUCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGUGAAUAUAUAUCACGGAUUUAAUC 139 GCCGUAUAAAAGCNAUGUCAAAGCCG Anti-EGFR aptamer UGCCGAUAUNNANCACGGAUUUAAUC 140 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGUCACUAAAUUGCACGUAUAUAAUC 141 GCCGUAGUAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAACCAUAAAGCACGUAAUAAAUC 142 GCCGUAUAUAAGCAUGUCaAAGCCG Anti-EGFR aptamer UGCCGCUAUAUAGCACGUAUUAAUCG 143 CCGUAGUAAAGCAUGUCaAAGCCG Anti-EGFR aptamer UGCCGCUAUAGCACACGGAAUUUAAU 144 CGCCGUAGUAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAGGUAUAUAACNCGGAUUUAAUC 145 GCCGUAGAAAAGCAUGUCNAAGCCG Anti-EGFR aptamer UGCUCCUAUAACACACGGAUUUAAUC 146 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGCCCGUAAUUGCACGGAUUUAAUCG 147 CCGUAGAAAAGCAUGUCCAAGCCGG Anti-EGFR aptamer ACUCCCUAUAUNGCAACUACAUAAUC 148 GCCGUAAAUAAGCAUGUNCAAGCCG Anti-EGFR aptamer UGAAGCUAGAUCACACUAAAUUAAUC 149 GCCGUAGAAAAGCAUGUCAAAAAAGC CG Anti-EGFR aptamer UGACUCUUUAUCCCCCGUACAUUAUU 150 cACCGACCAAAGCAUUACCAUCCCC Anti-EGFR aptamer UGACGCCCUAACACACGUAUAUAAUC 151 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGUCGCAAAAUAGCACGUAUUUAAUC 152 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGAGUGUAUAAUUCACGUAUUUAAUC 153 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCUACUAUAUCGUAGGUAACUAAUC 154 GCCCUACAAACUCACUCUAAAACCG Anti-EGFR aptamer UUACGCUAUAUCACACGGAAUUUUAA 155 UCGCCGUAGAAAAGCAUGUCCAAGCC G Anti-EGFR aptamer CCCAUCUGUACUACAGGAAUUUAAUC 156 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGCCCAUAAAUAGCACGGAUUUAAUC 157 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGCCGCAAUAACAUACACAUAUAAUC 158 GCCGUAGAAAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCAACUAUAUCGCACGUAUGUAAUC 159 GCCGUAGAAAAAGCAUGUCAAAGCC Anti-EGFR aptamer UUCCGCUAUAUAGCACGGAAUUAAUC 160 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UUCCGCUAAGUCACACGAAAUUAAUC 161 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UGUAGCAAUAUCACACGUAAUUAAUC 162 GCCGUAUAUAAGCAUGUCAAAGCCG Anti-EGFR aptamer UGCCGUUAUAUAUCACGGAUUUAAUC 163 GCCGUAGAAAAGCAUGUCCAAGCCG Anti-EGFR aptamer UAACACAUAUAUCAAGUAACUUAUCU 164 CCUUAGUAACCAUCUCCAAGCCG HLA-A*0201-restricted viral NLVPMVATV 178 peptide

TABLE 1B Description of Construct Sequences and SEQ ID NOs Description Sequence # Construct 6245 ELVMTQSPSSLTVTAGEKVIMSCKSS 165 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH(IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T(243))[8.8.14] MDYWGQGTTVTVSSGGGGSDVQLVQS (129-248)]-5-mer tetraglycyl-seryl GAEVKKPGASVKVSCKASGYTFTRYT linker (249-253)-scFv anti-CD3E MHWVRQAPGQGLEWIGYINPSRGYTN [humanized VH (Homo sapiens YADSVKGRFTITTDKSTSTAYMELSS IGHV1-46*01 (82.50%)-(IGHD)- LRSEDTATYYCARYYDDHYCLDYWGQ IGHJ6*01)[8.8.12](254-372)-18- GTTVTVSSGEGTSTGSGGSGGSGGAD mer linker (373-390)-V-KAPPA DIVLTQSPATLSLSPGERATLSCRAS (Mus musculus IGKV4-59*01 QSVSYMNWYQQKPGKAPKRWIYDTSK (81.70%)-IGKJ1*01 L124 > V (493) VASGVPARFSGSGSGTDYSLTINSLE [5.3.9](391-496)]-hexahistidine AEDAATYYCQQWSSNPLTFGGGTKVE (497-502) IKHHHHHH CAS Registry Number 1005198-65- 1 ChemID: 1005198-65-1 Construct 6246 ELVMTQSPSSLTVTAGEKVIMSCKSS 166 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH(IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T(243))[8.8.14] MDYWGQGTTVTVSSGGGGSDVQLVQS (129-248)]-5-mer tetraglycyl-seryl GAEVKKPGASVKVSCKASGYTFTRYT linker (249-253)-scFv anti-CD3E MHWVRQAPGQGLEWIGYINPSRGYTN [humanized VH (Homo sapiens YADSVKGRFTITTDKSTSTAYMELSS IGHV1-46*01 (82.50%)-(IGHD)- LRSEDTATYYCARYYDDHYCLDYWGQ IGHJ6*01)[8.8.12](254-372)-18- GTTVTVSSGEGTSTGSGGSGGSGGAD mer linker (373-390 -hexahistidine HHHHHH (391-396) Construct 6247 ELVMTQSPSSLTVTAGEKVIMSCKSS 167 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH(IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T (243))[8.8.14] MDYWGQGTTVTVSSGGGGSDIVLTQS (129-248)]-5-mer tetraglycyl-seryl PATLSLSPGERATLSCRASQSVSYMN linker (249-253)-anti-CD3E-V- WYQQKPGKAPKRWIYDTSKVASGVPA KAPPA (Mus musculus IGKV4- RFSGSGSGTDYSLTINSLEAEDAATY 59*01 (81.70%)-IGKJ1*01 L124 > V YCQQWSSNPLTFGGGTKVEIKHHHHH (356) H [5.3.9](254-359)1-hexahistidine (360-365) Construct 6248 ELVMTQSPSSLTVTAGEKVIMSCKSS 168 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH(IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T (243))[8.8.14] MDYWGQGTTVTVSSGGGGSDVQLVQS (129-248)]-5-mer tetraglycyl-seryl GAEVKKPGASVKVSCKASGYTFTRYT linker (249-253)-scFv anti-CD3E MHWVRQAPGQGLEWIGYINPSRGYTN [humanized VH (Homo sapiens YADSVKGRFTITTDKSTSTAYMELSS IGHV1-46*01 (82.50%)-(IGHD)- LRSEDTATYYCARYYDDHYCLDYWGQ IGHJ6*01)[8.8.12](254-372)-25- GTTVTVSSGEGTSTGSGAIPVSLRGS mer linker (373-397 containing GGSGGADDIVLTQSPATLSLSPGERA MMP2 cleavage site AIPVSLR TLSCRASQSVSSSYLAWYQQKPGQAP (SEQ ID NO: 46))-V-KAPPA RLLIYGASSRATGVPARFSGSGSGTD (Homo sapiens V-KAPPA from FTLTISSLEPEDFATYYCLQIYNMPI gantenerumab, CAS: 1043556-46-2; TFGQGTKVEIKHHHHHH 398-505); 87-hexahistidine (506- 511) Construct 6249 ELVMTQSPSSLTVTAGEKVTMSCKSS 169 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH (IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T(243))[8.8.14] MDYWGQGTTVTVSSGGGGSDIVLTQS (129-248)]-5-mer tetraglycyl-seryl PATLSLSPGERATLSCRASQSVSYMN linker (249-253)-scFv anti-CD3E WYQQKPGKAPKRWIYDTSKVASGVPA V-KAPPA (Mus musculus IGKV4- RFSGSGSGTDYSLTINSLEAEDAATY 59*01 (81.70%)-IGKJ1*01 L124 > V YCQQWSSNPLTFGGGTKVEIKGEGTS (493)[5.3.9](254-359)]- TGSGAIPVSLRGSGGSGGADDVQLVQ 25-mer linker (360-384 containing SGAEVKKPGASVKVSCKASGYTFTGY MMP2 cleavage site AIPVSLR YMHWVRQAPGQGLEWMGWINPNSGGT (SEQ ID NO: 46))-Ig heavy chain NYAQKFQGRVTITRDTSASTAYMELS V region (clone alpha-MUC1-1, SLRSEDTAVYYCARDFLSGYLDYWGQ GenBank Accession S36265; 385- GTLVTVSSHHHHHH 502)-hexahistidine (503-508) Construct 9327 ELVMTQSPSSLTVTAGEKVTMSCKSS 170 EpCAM V_(L)V_(H)-V_(H)DV_(L) QSLLNSGNQKNYLTWYQQKPGQPPKL single chain; scFv anti-EPCAM LIYWASTRESGVPDRFTGSGSGTDFT [Mus musculus V-KAPPA LTISSVQAEDLAVYYCQNDYSYPLTF (IGKV8-19*01 GAGTKLEIKGGGGSGGGGSGGGGSEV (98.00%)-IGKJ5*01 L126 > I(112)) QLLEQSGAELVRPGTSVKISCKASGY [12.3.9](1-113)-15-mer AFTNYWLGWVKQRPGHGLEWIGDIFP tris(tetraglycyl-seryl) linker (114- GSGNIHYNEKFKGKATLTADKSSSTA 128)-Mus musculus VH (IGHV1- YMQLSSLTFEDSAVYFCARLRNWDEP 54*01 (85.90%)-(IGHD)- MDYWGQGTTVTVSSGGGGSDVQLVQS IGHJ4*01, 5123 > T(243))[8.8.14] GAEVKKPGASVKVSCKASGYTFTRYT (129-248)]-5-mer tetraglycyl-seryl MHWVRQAPGQGLEWIGYINPSRGYTN linker (249-253)-scFv anti-CD3E YADSVKGRFTITTDKSTSTAYMELSS [humanized VH (Homo sapiens LRSEDTATYYCARYYDDHYCLDYWGQ IGHV1-46*01 (82.50%)-(IGHD)- GTTVTVSSGEGTSTGSGGGGSGGGGS IGHJ6*01)[8.8.12](254-372)-25- GGSGGADDIVLTQSPATLSLSPGERA mer linker (373-397)-V-KAPPA TLSCRASQSVSSSYLAWYQQKPGQAP (Homo sapiens V-KAPPA from RLLIYGASSRATGVPARFSGSGSGTD gantenerumab, CAS: 1043556-46-2; FTLTISSLEPEDFATYYCLQIYNMPI 398-505);-hexahistidine (506-511) TFGQGTKVEIKHHHHHH Anti-EpCAM sequence from Brischwein K et al, Mol. Immunol. (2006) 43: 1129-43 Construct 9328 ELVMTQSPSSLTVTAGEKVTMSCKSS 171 single chain; scFv anti-EPCAM QSLLNSGNQKNYLTWYQQKPGQPPKL [Mus musculus V-KAPPA LIYWASTRESGVPDRFTGSGSGTDFT (IGKV8-19*01 LTISSVQAEDLAVYYCQNDYSYPLTF (98.00%)-IGKJ5*01 L126 > I(112)) GAGTKLEIKGGGGSGGGGSGGGGSEV [12.3.9](1-113)-15-mer QLLEQSGAELVRPGTSVKISCKASGY tris(tetraglycyl-seryl) linker (114- AFTNYWLGWVKQRPGHGLEWIGDIFP 128)-Mus musculus VH (IGHV1- GSGNIHYNEKFKGKATLTADKSSSTA 54*01 (85.90%)-(IGHD)- YMQLSSLTFEDSAVYFCARLRNWDEP IGHJ4*01, S123 > T(243))[8.8.14] MDYWGQGTTVTVSSGGGGSDIVLTQS (129-248)]-5-mer tetraglycyl-seryl PATLSLSPGERATLSCRASQSVSYMN linker (249-253)-scFv anti-CD3E WYQQKPGKAPKRWIYDTSKVASGVPA V-KAPPA (Mus musculus IGKV4- RFSGSGSGTDYSLTINSLEAEDAATY 59*01 (81.70%)-IGKJ1*01 L124 > V YCQQWSSNPLTFGGGTKVEIKGEGTS (493)[5.3.9](254-359)]-25-mer TGSGGGGSGGGGSGGSGGADDVQLVQ linker (360-384)-Ig heavy chain V SGAEVKKPGASVKVSCKASGYTFTGY region (clone alpha-MUC1-1, YMHWVRQAPGQGLEWMGWINPNSGGT GenBank Accession S36265; 385- NYAQKFQGRVTITRDTSASTAYMELS 502)-hexahistidine (503-508) SLRSEDTAVYYCARDFLSGYLDYWGQ GTLVTVSSHHHHHH Construct 9329 QVQLVQSGGGLVQPGGSLRLSCAASY 172 Glypican3 V_(HH)-CD3ε(V_(H)-MMP2- FDFDSYEMSWVRQAPGKGLEWIGSIY V_(L)) HSGSTYYNPSLKSRVTISRDNSKNTL Anti-human Glypican-3 VHH YLQMNTLRAEDTATYYCARVNMDRFD sequence from U.S. Pat. No. YWGQGTLVTVSSSGGGGSDVQLVQSG 2012145469; residues 1-116)-5-mer AEVKKPGASVKVSCKASGYTFTRYTM tris(tetraglycyl-seryl) linker (117- HWVRQAPGQGLEWIGYINPSRGYTNY 122)- ADSVKGRFTITTDKSTSTAYMELSSL scFv anti-CD3E RSEDTATYYCARYYDDHYCLDYWGQG [humanized VH (Homo sapiens TTVTVSSGEGTSTGSGAIPVSLRGSG IGHV1-46*01 (82.50%)-(IGHD)- GSGGADDIVLTQSPATLSLSPGERAT IGHJ6*01) [8.8.12](123-241)-25- LSCRASQSVSSSYLAWYQQKPGQAPR mer linker (242-266 containing LLIYGASSRATGVPARFSGSGSGTDF MMP2 cleavage site AIPVSLR TLTISSLEPEDFATYYCLQIYNMPIT (SEQ ID NO: 46))-V-KAPPA FGQGTKVEIKHHHHHH (Homo sapiens V-KAPPA from gantenerumab, CAS: 1043556-46-2; 267-374); -hexahistidine (375-380) Construct 9330 DIQMTQSTSSLSASLGDRVTISCSAS 173 anti-[Homo sapiens SDC1 QGINNYLNWYQQKPDGTVELLIYYTS (syndecan-1, CD138), scFv, from TLQSGVPSRFSGSGSGTDYSLTISNL indatuximab CAS: 1238517-16-2, EPEDIGTYYCQQYSKLPRTFGGGTKL U.S. Pat. No. U520140010828], [Mus EIKRGGGGSGGGGSGGGGSQVQLQQS musculus V-KAPPA (IGKV10- GSELMMPGASVKISCKATGYTFSNYW 94*01-IGKJ1*01)[6.3.9](1-108) - IEWVKQRPGHGLEWIGEILPGTGRTI 15-mer tris(tetraglycyl-seryl) linker YNEKFKGKATFTADISSNTVQMQLSS (109-123) [Mus musculus VH LTSEDSAVYYCARRDYYGNFYYAMDY (IGHV1-9*01 -(IGHD)- WGQGTSVTVSSGGGGSDVQLVQSGAE IGHJ4*01)[8.8.15](124-245)-5- VKKPGASVKVSCKASGYTFTRYTMHW mer tris(tetraglycyl-seryl) linker VRQAPGQGLEWIGYINPSRGYTNYAD (246-250)-scFv anti-CD3E SVKGRFTITTDKSTSTAYMELSSLRS [humanized VH (Homo sapiens EDTATYYCARYYDDHYCLDYWGQGTT IGHV1-46*01 (82.50%)-(IGHD)- VTVSSGEGTSTGSGAIPVSLRGSGGS IGHJ6*01)[8.8.12](251-369)-25- GGADDIVLTQSPATLSLSPGERATLS mer linker (370-394 containing CRASQSVSSSYLAWYQQKPGQAPRLL MMP2 cleavage site AIPVSLR IYGASSRATGVPARFSGSGSGTDFTL (SEQ ID NO: 46))-V-KAPPA TISSLEPEDFATYYCLQIYNMPITFG (Homo sapiens V-KAPPA from QGTKVEIKHHHHHH gantenerumab, CAS: 1043556-46-2; 395-502); -hexahistidine (503-508) Construct 9332 QVKLEESGGGSVQTGGSLRLTCAASG 174 EGFR V_(HH)-CD3ε(V_(H)-V_(L)) RTSRSYGMGWFRQAPGKEREFVSGIS Anti-human EGFR V_(HH) sequence WRGDSTGYADSVKGRFTISRDNAKNT from 7D12 sequence from Schmitz VDLQMNSLKPEDTAIYYCAAAAGSAW KR et al, Structure. 2013 July YGTLYEYDYWGQGTQVTVSSGGGGSG 2; 21(7): 1214-24; residues 1-124)- GGGSGGGGSGGGGSGGGGSGGGGSDV 30-mer tris(tetraglycyl-seryl) linker QLVQSGAEVKKPGASVKVSCKASGYT (125-154)-scFv anti-CD3E FTRYTMHWVRQAPGQGLEWIGYINPS [humanized VH (Homo sapiens RGYTNYADSVKGRFTITTDKSTSTAY IGHV1-46*01 (82.50%)-(IGHD)- MELSSLRSEDTATYYCARYYDDHYCL IGHJ6*01)[8.8.12](155-273)-18- DYWGQGTTVTVSSGEGTSTGSGGSGG mer linker (274-291) -V-KAPPA SGGADDIVLTQSPATLSLSPGERATL (Mus musculus IGKV4-59*01 SCRASQSVSYMNWYQQKPGKAPKRWI (81.70%)-IGKJ1*01 L124 > V (394) YDTSKVASGVPARFSGSGSGTDYSLT [5.3.9](292-397)]-hexahistidine INSLEAEDAATYYCQQWSSNPLTFGG (398-403) GTKVEIKHHHHHH Anti-human CD3ε sequence from Brischwein K et al, Mol. Immunol. (2006) 43: 1129-43 U.S. Pat. No. US7919089 Construct 9333 EVQLVESGGGLVQAGGSLRLSCAASG 175 EGFR V_(HH)-CD3ε(V_(H)-V_(L)) RTFSSYAMGWFRQAPGKEREFVVAIN Anti-human EGFR V_(HH) sequence WSSGSTYYADSVKGRFTISRDNAKNT from 9G8 sequence from Schmitz MYLQMNSLKPEDTAVYYCAAGYQINS KR et al, Structure. 2013 July GNYNFKDYEYDYWGQGTQVTVSSGGG 2; 21(7): 1214-24; residues 1-127)- GSGGGGSGGGGSGGGGSGGGGSGGGG 30-mer tris(tetraglycyl-seryl) linker SDVQLVQSGAEVKKPGASVKVSCKAS (128-157)-scFv anti-CD3E GYTFTRYTMHWVRQAPGQGLEWIGYI [humanized VH (Homo sapiens NPSRGYTNYADSVKGRFTITTDKSTS IGHV1-46*01 (82.50%)-(IGHD)- TAYMELSSLRSEDTATYYCARYYDDH IGHJ6*01)[8.8.12](158-276)-18- YCLDYWGQGTTVTVSSGEGTSTGSGG mer linker (277-294)-V-KAPPA SGGSGGADDIVLTQSPATLSLSPGER (Mus musculus IGKV4-59*01 ATLSCRASQSVSYMNWYQQKPGKAPK (81.70%)-IGKJ1*01 L124 > V (394) RWIYDTSKVASGVPARFSGSGSGTDY [5.3.9](292-397)]-hexahistidine SLTINSLEAEDAATYYCQQWSSNPLT (401-406) FGGGTKVEIKHHHHHH Construct 9334 QVKLEESGGGSVQTGGSLRLTCAASG 176 EGFR V_(HH)-CD3ε(V_(H)-MMP2-V_(L)) RTSRSYGMGWFRQAPGKEREFVSGIS Anti-human EGFR V_(HH) sequence WRGDSTGYADSVKGRFTISRDNAKNT from 7D12 sequence from Schmitz VDLQMNSLKPEDTAIYYCAAAAGSAW KR et al, Structure. 2013 July YGTLYEYDYWGQGTQVTVSSGGGGSG 2; 21(7): 1214-24; residues 1-124)- GGGSGGGGSGGGGSGGGGSGGGGSDV 30-mer tris(tetraglycyl-seryl) linker QLVQSGAEVKKPGASVKVSCKASGYT (125-154)- FTRYTMHWVRQAPGQGLEWIGYINPS scFv anti-CD3E RGYTNYADSVKGRFTITTDKSTSTAY [humanized VH (Homo sapiens MELSSLRSEDTATYYCARYYDDHYCL IGHV1-46*01 (82.50%)-(IGHD)- DYWGQGTTVTVSSGEGTSTGSGAIPV IGHJ6*01)[8.8.12](155-273)-25- SLRGSGGSGGADDIVLTQSPATLSLS mer linker (274-298 containing PGERATLSCRASQSVSSSYLAWYQQK MMP2 cleavage site AIPVSLR PGQAPRLLIYGASSRATGVPARFSGS (SEQ ID NO: 46))-V-KAPPA GSGTDFTLTISSLEPEDFATYYCLQI (Homo sapiens V-KAPPA from YNMPITFGQGTKVEIKHHHHHH gantenerumab, CAS: 1043556-46-2; 299-406); -hexahistidine (407-412) Construct 9335 EVQLVESGGGLVQAGGSLRLSCAASG 177 Anti-human EGFR V_(HH) sequence RTFSSYAMGWFRQAPGKEREFVVAIN from 9G8 sequence from Schmitz WSSGSTYYADSVKGRFTISRDNAKNT KR et al, Structure. 2013 July MYLQMNSLKPEDTAVYYCAAGYQINS 2; 21(7): 1214-24; residues 1-127)- GNYNFKDYEYDYWGQGTQVTVSSGGG 30-mer tris(tetraglycyl-seryl) linker GSGGGGSGGGGSGGGGSGGGGSGGGG (128-157)- SDIVLTQSPATLSLSPGERATLSCRA scFv anti-CD3E SQSVSYMNWYQQKPGKAPKRWIYDTS V-KAPPA (Mus musculus IGKV4- KVASGVPARFSGSGSGTDYSLTINSL 59*01 (81.70%)-IGKJ1*01 L124 > V EAEDAATYYCQQWSSNPLTFGGGTKV (493)[5.3.9](158-263)]-25-mer EIKGEGTSTGSGAIPVSLRGSGGSGG linker (264-288 containing MMP2 ADDVQLVQSGAEVKKPGASVKVSCKA cleavage site AIPVSLR (SEQ ID SGYTFTGYYMHWVRQAPGQGLEWMGW NO: 46))-Ig heavy chain V region INPNSGGTNYAQKFQGRVTITRDTSA (clone alpha-MUC1-1, GenBank STAYMELSSLRSEDTAVYYCARDFLS Accession S36265; 289-406)- GYLDYWGQGTLVTVSSHHHHHH hexahistidine (307-412)

DESCRIPTION OF THE EMBODIMENTS

I. A Two-Component System Comprising at Least One Targeted T-Cell Engaging Agent

A variety of targeted T-cell engaging agents are described in different embodiments, and in some embodiment as part of a two-component system comprising a first component and a second component. In each of the embodiments, however, a targeting moiety may be used to deliver the targeted T-cell engaging agent to an area of unwanted cells, allowing for a therapeutic effect to be delivered locally. The targeted T-cell engaging agent also contains a first T-cell engaging domain capable of activity when binding a second T-cell engaging domain, but the second T-cell engaging domain is not part of the targeted T-cell engaging agent. In other words, without the second T-cell engaging domain that is not part of the targeted T-cell engaging agent, the first T-cell engaging domain is not capable of T-cell engaging activity. The targeted T-cell engaging agent also comprises an inert binding partner capable of binding the first T-cell engaging domain and preventing it from binding to a second T-cell engaging domain. In other words, the inert binding partner binds to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed. By does not bind, the application does not exclude nonspecific binding or low levels of binding (for example, ≤1%, ≤5%, ≤10%). The concept is one of functional insufficiency with the de novo VH/VL complementation insufficient for T-cell target binding. Proteolytic cleavage liberates the inert VH or VL groups allowing the opportunity for re-pairing of active VH and VL pairs at the cell surface. Furthermore, the targeted T-cell engaging agent includes a cleavage site separating the first T-cell engaging domain and the inert binding partner. The cleavage site is cleaved when the targeted T-cell engaging agent is in the microenvironment of the unwanted cells.

In some embodiments, the second T-cell engaging domain is part of a second targeted T-cell engaging agent. Thus, in some embodiments, a kit or composition may comprise two targeted T-cell engaging agents, one with a first T-cell engaging domain and another with a second T-cell engaging domain. In such a kit or composition, the inert binding partners may be capable of dissociation once the cleavage site in each agent has been cleaved; after dissociation, the two T-cell engaging domains may be capable of binding to each other and exhibiting activity.

In some embodiments with two targeted T-cell engaging agents, the two-component system comprises one T-cell engaging domain that may be a VH domain and another T-cell engaging domain that may be a VL domain. In embodiments with two targeted T-cell engaging agents, the targeting moieties in the first component and the second component may be the same or they may be different.

In embodiments with two targeted T-cell engaging agents, the cleavage sites in the first component and the second component may be the same or they may be different.

FIG. 1 shows one embodiment of a targeted T-cell engaging agent construct comprising (a) an scFv targeting domain comprising a VH domain and a VL domain that bind the target, wherein the VH and VL domain are connected by a flexible linker; (b) an inactive T-cell engaging domain comprising a VL domain that binds to an inert VH domain, wherein the VH and VL domains are connected by a flexible linker having a cleavage site, and (c) a flexible linker joining the targeting domain and the inactive T-cell engaging domain.

In some embodiments, FIG. 2 shows the process by which the cleavable linker is cleaved and the inert binding partner is released to create an entity without an inert binding partner. This entity is still inactive because the VL domain in the T-cell engaging domain is not active on its own.

In some embodiments, FIG. 3 illustrates the creation of an active targeted T-cell engaging agent after the inert binding partner is released from a pair of complementary targeted T-cell engaging agents.

In some embodiments, FIGS. 4A-C illustrate the cleavage of the stepwise process of the targeted T-cell engaging agents binding to the target cell (4A), cleavage of the inert binding partners (4A and 4B), and binding to create an active targeted T-cell engaging agent (4C).

In some alternative embodiments, the second T-cell engaging domain may not be bound to a targeting moiety and/or may not comprise a cleavage site and inert binding partner. In some instances, the second T-cell engaging domain may be conjugated or linked to a targeting moiety (either the same targeting moiety or a different targeting moiety), but in such an embodiment it would not be conjugated or linked to an inert binding partner. In such an embodiment, only the first T-cell engaging domain is bound to an inert binding partner. In another embodiment, the second T-cell engaging domain may also comprise a targeting moiety, a cleavage site, and an inert binding partner, each as described herein.

In some embodiments, the structural arrangement from N-terminus to C-terminus of the first component comprises IBVL-L1-TCEVH-L2-TVL-L3-TVH. In some embodiments, the structural arrangement from N-terminus to C-terminus of the second component comprises TCEVL-L2-TVH-L3-TVL. In some embodiments, the structural arrangement from N-terminus to C-terminus of the second component comprises IBVH-L1-TCEVL-L2-TBVH-L3-TBVL. In each of these embodiments IB stands for inert binding partner and IBVL is a VL inert binding partner, whereas IBVH is a VH insert binding domain. TCE stands for T-cell engaging and an TCEVL is a VL portion of a T-cell engaging domain and a TCEVH is a VH portion of a T-cell engaging domain. TB stand for target binding domain and a TBVH is a VH portion of a target binding domain and a TBVL is a VL portion of a target binding domain. L1 is a linker with a protease cleavage site, while L2 and L3 are optionally linkers that optionally are not cleavable by the same protease as L1.

A. Targeting Moiety

The targeting moiety functions in the targeted T-cell engaging agent by delivering the agent to the local environment of the unwanted cells, enabling a localized treatment strategy. In certain embodiments, the targeting moiety targets the unwanted cells by specifically binding to the unwanted cells. In some instances, the targeting moiety specifically binds the unwanted cells even while the inert binding partner is binding the first T-cell engaging domain.

In some embodiments, a first targeting moiety is bound, optionally by a linker, to a first T-cell engaging domain and, as part of a separate construct, a second targeting moiety is bound, optionally by a linker, to a second T-cell engaging domain. In this way, each complementary part of the T-cell engaging domain is delivered to the unwanted cells by a separate targeting moiety. In some embodiments, the targeting moieties are of the same type and, in some embodiments, the targeting moieties are different. When the targeting moieties are of different types, they can either target different epitopes (either overlapping or nonoverlapping) on the same target protein of the unwanted cell or they can target different target proteins. In situations when the targeting moieties target different proteins, the unwanted cell will express an antigen corresponding to each of the two types of targeting moieties, providing additional specificity for this approach.

In certain embodiments, the targeting moiety is an antibody or functional part thereof. By functional part, we mean any antibody fragment that retains its binding activity to the target on the unwanted cell, such as an scFv or VHH or other functional fragment including an immunoglobulin devoid of light chains, Fab, Fab′, F(ab′)₂, Fv, antibody fragment, diabody, scAB, single-domain heavy chain antibody, single-domain light chain antibody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. Unless specifically noted as “full length antibody,” when the application refers to antibody it inherently includes a reference to a functional part thereof.

Certain antibody targets (with examples of unwanted cell types in parentheses) may include: Her2/Neu (Epithelial malignancies); CD22 (B cells, autoimmune or malignant); EpCAM (CD326) (Epithelial malignancies); EGFR (epithelial malignancies); PMSA (Prostate Carcinoma); CD30 (B cell malignancies); CD20 (B cells, autoimmune, allergic or malignant); CD33 (Myeloid malignancies); membrane 1gE (Allergic B cells); 1gE Receptor (CD23) (Mast cells or B cells in allergic disease), CD80 (B cells, autoimmune, allergic or malignant); CD86 (B cells, autoimmune, allergic or malignant); CD2 (T cell or NK cell lymphomas); CA125 (multiple cancers including Ovarian carcinoma); Carbonic Anhydrase IX (multiple cancers including Renal Cell Carcinoma); CD70 (B cells, autoimmune, allergic or malignant); CD74 (B cells, autoimmune, allergic or malignant); CD56 (T cell or NK cell lymphomas); CD40 (B cells, autoimmune, allergic or malignant); CD19 (B cells, autoimmune, allergic or malignant); c-met/HGFR (Gastrointestinal tract and hepatic malignancies; TRAIL-R1 (multiple malignancies including ovarian and colorectal carcinoma); DRS (multiple malignancies including ovarian and colorectal carcinoma); PD-1 (B cells, autoimmune, allergic or malignant); PD1L (Multiple malignancies including epithelial adenocarcinoma); IGF-1R (Most malignancies including epithelial adenocarcinoma); VEGF-R2 (The vasculature associated with the majority of malignancies including epithelial adenocarcinomas; Prostate stem cell antigen (PSCA) (Prostate Adenocarcinoma); MUC1 (Epithelial malignancies); CanAg (tumors such as carcinomas of the colon and pancreas); Mesothelin (many tumors including mesothelioma and ovarian and pancreatic adenocarcinoma); P-cadherin (Epithelial malignancies, including breast adenocarcinoma); Myostatin (GDF8) (many tumors including sarcoma and ovarian and pancreatic adenocarcinoma); Cripto (TDGF1) (Epithelial malignancies including colon, breast, lung, ovarian, and pancreatic cancers); ACVRL 1/ALK1 (multiple malignancies including leukemias and lymphomas); MUC5AC (Epithelial malignancies, including breast adenocarcinoma); CEACAM (Epithelial malignancies, including breast adenocarcinoma); CD137 (B cells or T cells, autoimmune, allergic or malignant); CXCR4 (B cells or T cells, autoimmune, allergic or malignant); Neuropilin 1 (Epithelial malignancies, including lung cancer); Glypicans (multiple cancers including liver, brain and breast cancers); HER3/EGFR (Epithelial malignancies); PDGFRa (Epithelial malignancies); EphA2 (multiple cancers including neuroblastoma, melanoma, breast cancer, and small cell lung carcinoma); CD38 (Myeloma); CD138 (Myeloma); α4-integrin (AML, myeloma, CLL, and most lymphomas).

In certain modes, antibodies include an anti-epidermal growth factor receptor antibody such as Cetuximab, an anti-Her2 antibody, an anti-CD20 antibody such as Rituximab, an anti-CD22 antibody such as Inotuzumab, G544 or BU59, an anti-CD70 antibody, an anti-CD33 antibody such as hp67.6 or Gemtuzumab, an anti-MUC1 antibody such as GP1.4 and SM3, an anti-CD40 antibody, an anti-CD74 antibody, an anti-P-cadherin antibody, an anti-EpCAM antibody, an anti-CD138 antibody, an anti-E-cadherin antibody, an (anti-CEA antibody, an anti-FGFR3 antibody, and an anti α4-integrin antibody such as natalizumab.

Table 2A provides nonlimiting examples of cancer types, possible targeting moieties, and proteases that are expressed by those cancer types. In order to prepare a two-component system, the cancer may be identified from column 1, one or two targets chosen for the targeting moiety (as desired), and one or two proteases chosen for the cancer type, as well (as desired). Other sections of this application discuss when to use one versus two targeting moieties and one versus two protease cleavage sites.

TABLE 2A Coordination of Cancer Type, Targets for Targeting Moiety, and Proteases that Can Cleave Cleavage Sites Proteases that can Cleave Cancer Targets for Targeting Moiety Cleavage Site Prostate ADAM17, CD59, EpCAM, HER2, KLK3 (PSA), Cancer Integrin αV, Integrin αVβ3, MCP-1, PCLA, KLK4, ADAM17, PSCA, PSMA, RANKL, RG1, SLC44A4 Cathepsin B, uPA, STEAP-1, VEGF-C uPAR, HPN, ST14, TMPRSS2 Breast Cancer CA125, CCN1, CD44, CD98, c-RET, DLL4, MMP2, MMP9, EpCAM, Episialin, GPNMB, HER2/neu, Cathepsin L, HER3, IGF-1R, Integrin α6β4, LFL2, LIV- Cathepsin K, 1, Ly6E, MUC1, MUC18, NRP1, Cathepsin B, Phosphatidylserine, PRLR, TACSTD-2, MMP11, HPN, Tenascin C, TWEAKR, VANGL2, PD-L1, ST14, ADAM28 PD-L2 Myeloma BCMA, IGF-1R, DKK-1, ICAM-1, MMP2, MMP9, CD138/Syndecan1, CD38, GRP78, FGFR3, MMP1, MMP7, SLAMF6, CD48, TfR(CD71) APRIL, CD40, TMPRSS2, CD19, DR5, CXCR4 PRSS22, KLK11 B-cell Lymphoma CD20, CD22, CD19, CD37, CD70, HLA- ADAM28, DR, CD70b Cathepsin B, MMP9 Renal Cell PD-L, PD-L2, CAIX, TPBG, CD70, ST14, MMP9 carcinoma ENPP3, FGFR1 Gastric VEGFR-2, CLDN18, GCC, C242, MMP2, MMP9, Carcinoma HER2/neu, FGFR2, EpCAM, GPR49, Cathepsin B, uPA, HER3, IGFR uPAR Glioblastoma HER2/neu, EGFR, ALK, EphA2, GD2, MMP2, MMP9, EGFRvIII, ALK T-cell CD2, CD4, CD5, CD71, CD30 Cathepsin B, lymphoma Cathepsin D, MMP9 Hodgkin CD30, CD40, IL-3Ra, CD30 Cathepsin B Lymphoma Lung Cancer EGFR, IGF-1R, HER3, Integrin α5β1, Cathepsin B, Lewis y/b antigen, EGFL7, TPBG, DKK-1, MMP2, MMP9, NaPi2b, flt4, cMet, CD71 ST14, ADAM17 Pancreatic SLC44A4, uPAR, MUC1, MUCH16, Cathepsin B, ST14, Carcinoma TACSTD-2, CEA, EphhA4, mesothelin, ADAM28 EGFR, MUC13, MU5AC, AGF-1R, HER3, CD71 Head and EGFR, EpCAM, HER2 Cathepsin B, ST14, Neck cancer ADAM17 Acute myeloid CD33, CD133, CD123, CD45, CD98, c-Kit, ADAM17, leukemia Lewis Y, Siglec-15, FLT-3 Cathepsin B, uPA, uPAR Melanoma MUC18, CD40, GD2, CEACAM1, Cathepsin B, Cadherin-19, GM3, Integrin α5β1, TYRP1, MMP9 GD3, Integrin αV Ovarian HER2/neu, EpCAM, CA125, DLL4, Cathepsin B, Cancer Integrin αVβ3, MUC5A, NaPi2B, MMP2, MMP9 Mesothelin, CLDN6 Liver Cancer Glypican-3, FGFR4, ENPP3, PIVKA-II, Cathepsin B, PLVAP, cMet, EpCAM MMP9 Colorectal EGFR, Lewis y/b, Progastrin, GPR49, CEA, Cathepsin S, Carcinoma CLDN1, A33, CK8, Integrin αV, EpCAM, Cathepsin L, DLL4, EGFL7, FAP, Cathepsin B, uPA, uPAR, MMP2, MMP9, ST14

For example, when targeting moieties in the first and second components are different, Table 2B provides a nonlimiting list of potential targeting moieties to use in combination with particular cancer types. In a two-component system, a targeting moiety for the first component would be present and a second targeting moiety for the second component may optionally be present. If only the first component has a targeting moiety or if the first and second components have the same targeting moiety, either the targeting moiety listed in column 1 or column 2 of the table may be used when the cancer type is listed in column 3.

TABLE 2B Targeting Moieties for Use in Two-Component System Optional Targeting Targeting Moiety for Moiety for Second First Component Component Cancer Type Antibody against CD20 Antibody against CD80 Lymphoma (such as Rituximab) Antibody against CD20 Antibody against CD22 Lymphoma (such as Rituximab) (such as Inotuzumab) Antibody against CD20 Antibody against CD70 Lymphoma (such as Rituximab) Antibody against HER2 Antibody against EpCAM Epithelial malignancies Antibody against EGFR Antibody against mucin Breast cancer (such as Cetuximab) protein core Antibody against EGFR Antibody against HER2 Epithelial malignancies (such as Cetuximab) Antibody against EGFR Antibody against Gliomas (such as Cetuximab) transferrin receptor Antibody against Antibody against p- Drug-resistant melanomas gp95/gp97 glycoprotein Antibody against TRAIL- Antibody against DR5 Multiple malignancies, R1 including ovarian and colorectal carcinoma Antibody against IL-4 Antibody against IL-6 Lymphomas and leukemias Antibody against CD19 Antibody against CD22 Lymphoma Antibody against PMSA Antibody against PSCA Prostate carcinoma Antibody against P- Antibody against Cripto Epithelial malignancies cadherin (TDGF1) Antibody against CD74 Antibody against CD40 Lymphomas Antibody against PD1L Antibody against IGF-1R Epithelial adenocarcinoma Antibody against CD38 Antibody against CD138 Myeloma Antibody against BCMA Antibody against CD138 Myeloma or CD38 Antibody against CD33 Antibody against CD133 Myeloid Malignancies, e.g. AML Antibody against CD33 Antibody against CD123 Myeloid Malignancies such as AML Antibody against CD49d Antibody against CD33 Myeloid Malignancies Antibody against PSMA Antibody against PSCA Prostate Cancer Antibody against Antibody against cMet or Hepatocellular carcinoma Glypican 3 EpCAM Antibody against EpCAM Antibody against EGFR Lung Cancer Antibody against EpCAM Antibody against MUC1 Pancreatic Cancer Antibody against EpCAM Antibody against EGFR Colorectal Carcinoma Antibody against MUC1 Antibody against EGFR Ovarian Carcinoma Antibody against GD2 Antibody against HER2 Sarcoma Antibody against HER2 Antibody against HER3 Breast Cancer Antibody against IL-13R Antibody against EGFR Brain Cancer

In some embodiments, the targeting moiety is not an antibody, but is another type of targeting moiety. For example, a targeting moiety may be a binding partner for a protein known to be expressed on the unwanted cell. Such expression levels may include overexpression. For example, the following binding partners may bind to the following targets on an unwanted cell:

TABLE 3 Non-Antibody Binding Partners and Corresponding Targets Binding Partner Target on Unwanted Cell IL-2 IL-2 receptor IL-4 IL-4 receptor IL-6 IL-6 receptor α-MSH MSH receptor (melanocyte stimulating hormone receptor) Transferrin TR (transferrin receptor) Folic acid FOLR (folate receptor 1) and/or FOLH1 (folate hydroxylase) EGF and/or TGFα EGFR (EGF receptor) PD1 PD-L1 and/or PD-L2 IL13 IL-13R (Glioblastoma) Stem cell factor CXCR4 Insulin-like growth factor (IGF) IGFR CD40 CD40L

The binding partner need not comprise the full length or wildtype sequence for the binding partners listed in Table 3. All that is required is that the binding partner bind to the target on the unwanted cell and can thus include truncated forms, analogs, variants, and derivatives that are well known in the art.

Additionally, in some embodiments, the binding partner may be an aptamer that is capable of binding to a protein known to be expressed on the unwanted cell. Aptamers that bind unwanted cells, such as cancer cells, are well known and methods for designing them are known.

Cell-based SELEX systems may be used to select a panel of target cell-specific aptamers from a random candidate library. A ssDNA pool may be dissolved in binding buffer and denatured and then incubated with target cells. After washing the bound DNAs may be eluted by heating and then incubated with negative cells (if desired), centrifuged, and the supernatant removed. The supernatant may be amplified by PCR with biotin labeled primers. The selected sense ssDNA may be separated from the antisense biotinylated strand using streptavidin coated beads. To increase affinity, washing strength may be increased through increasing washing time, volume of buffer, and number of washes. After the desired rounds of selection, the selected ssDNA pool may be PCR amplified and cloned into E. coli and sequenced. See Shangguan et al., Aptamers evolved from live cells as effective molecular probes for cancer study, PNAS 103(32:11838-11843 (2006); Lyu et al, Generating Cell Targeting Aptamers for Nanotherapeutics Using Cell-SELEX, Theranostics 6(9):1440-1452 (2016); see also Li et al., Inhibition of Cell Proliferation by an Anti-EGFR Aptamer, PLoS One 6(6):e20229 (2011). The specific approaches for designing aptamers and specific aptamers binding to cancer cells in these references are hereby incorporated by reference.

For example, an aptamer may comprise SEQ ID NO: 94 to 164. In some embodiments, an aptamer may comprise SEQ ID NO: 95. These aptamers are directed to EGFR and are provided only as representative of the aptamers that can bind to targets presented on unwanted cells. Other aptamers against other targets on unwanted cells are equally part of the description herein and incorporated by reference as described in Zhu et al., Progress in Aptamer Mediated Drug Delivery Vehicles for Cancer Targeting, Theranostics 4(9):931-944 (2014).

In some embodiments, aptamers for use herein bind to the target on the unwanted cell with a K_(d) in the nanomolar to picomolar range (such as 1 picomolar to 500 nanomolar or 1 picomolar to 100 nanomolar).

B. T-Cell Engaging Domain

The targeted T-cell engaging agent comprises a first T-cell engaging domain that is unable of engaging a T-cell alone. Instead, the first T-cell engaging domain is capable of activity when binding a second T-cell engaging domain, which is not part of the targeted T-cell engaging agent. Thus, the first and second T-cell engaging domains may be any two moieties that do not possess T-cell engaging activity alone, but do possess it when paired with each other. In other words, the first and second T-cell engaging domains are complementary halves of a functional active protein.

When the two T-cell engaging domains are associated together in the two-component system, they may bind to the CD3 antigen and/or T-cell receptor on the surface of the T-cell as these activate T cells. CD3 is present on all T cells and consists of subunits designated γ, δ, ε, ζ, and η. The cytoplasmic tail of CD3 is sufficient to transduce the signals necessary for T cell activation in the absence of the other components of the TCR receptor complex. Normally, activation of T cell cytotoxicity depends first on binding of the TCR with a major histocompatibility complex (MHC) protein, itself bound to a foreign antigen, located on a separate cell. In a normal situation, only when this initial TCR-MHC binding has taken place can the CD3 dependent signally cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue. In some of the present embodiments, however, when the two-component system binds to CD3 and/or the TCR, activation of cytotoxic T cells in the absence of independent TCR-MHC can take place by virtue of the crosslinking of the CD3 and/or TCR molecules mimicking an immune synapse formation. This means that T cells may be cytotoxically activated in a clonally independent fashion, i.e. in a manner that is independent of the specific TCR clone carried by the T cell. This allows for activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.

In some embodiments, the first T-cell engaging domain is a VH domain and the second T-cell engaging domain is a VL domain. In other embodiments, the first T-cell engaging domain is a VL domain and the second T-cell engaging domain is a VH domain. In such embodiments, when paired together the first and second T-cell engaging domains may comprise an scFv.

If the first and second T-cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a T cell, such as CD3 or TCR. If the antigen is CD3, one potential T-cell engaging domain may be derived from muromonab.

C. Inert Binding Partner

The targeted T-cell engaging agent also comprises at least one inert binding partner capable of binding the first T-cell engaging domain and preventing it from binding to a second T-cell engaging domain unless certain conditions occur. When the first T-cell engaging domain is bound to the at least one inert binding partner, it does not possess a T-cell engaging activity. In other words, the at least one inert binding partner cripples the function of the first T-cell engaging domain by blocking it from binding its complementary pair (the second T-cell engaging domain) and preventing the two domains from joining together to have a T-cell engaging activity. In other words, the inert binding partner binds to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed. By does not bind, the application does not exclude nonspecific binding or low levels of binding (for example, ≤1%, ≤5%, ≤10%).

In some embodiments, the inert binding partner binds specifically to the T-cell engaging domain.

In some embodiments, the at least one inert binding partner is a VH or VL domain. In some embodiments, when the T-cell engaging domain in the targeted T-cell engaging agent is a VH domain, the inert binding partner may be a VL domain and when the first T-cell engaging domain is a VL domain, the inert binding partner may be a VH domain.

If a first component comprises a targeting moiety and a VL T-cell engaging domain and a VH inert binding partner, in some embodiments, the VH inert binding partner has an equilibrium dissociation constant for binding to the VL T-cell engaging domain, which is greater than the equilibrium dissociation constant of the VL T-cell engaging domain for its partner VH T-cell engaging domain in the second component. In some embodiments, the prior sentence is equally true when VH is switched for VL and vice versa.

Based on empirical evidence in the examples, it is believed that using the inert binding partner as a mispairing partner with the T-cell engaging domain in the construct results in constructs that are more stable and easier to manufacture.

D. Cleavage Site

By way of overview, the cleavage site may be (i) cleaved by an enzyme expressed by the unwanted cells; (ii) cleaved through a pH-sensitive cleavage reaction inside the unwanted cell; (iii) cleaved by a complement-dependent cleavage reaction; or (iv) cleaved by a protease that is colocalized to the unwanted cell by a targeting moiety that is the same or different from the targeting moiety in the agent. In some embodiments, the cleavage site is a protease cleavage site.

The cleavage sites function to release the inert binding partner from the first T-cell engaging domain. The cleavage sites can function in different ways to release the inert binding partner from the first T-cell engaging domain T-cell epitopes in the microenvironment of the unwanted cells. The cleavage may occur inside the unwanted cell or outside the unwanted cell, depending on the strategy employed. If cleavage occurs outside the unwanted cell, the T-cell engaging domain can be presented without first being internalized into a cell and being engaged in the classical antigen-processing pathways.

In certain embodiments, at least one cleavage site may be cleaved by an enzyme expressed by the unwanted cells. Cancer cells, for instance, are known to express certain enzymes, such as proteases, and these may be employed in this strategy to cleave the targeted T-cell engaging agent's cleavage site. By way of nonlimiting example, cathepsin B cleaves FR, FK, VA and VR amongst others; cathepsin D cleaves PRSFFRLGK (SEQ ID NO: 42), ADAM28 cleaves KPAKFFRL (SEQ ID NO: 1), DPAKFFRL (SEQ ID NO: 2), KPMKFFRL (SEQ ID NO: 3) and LPAKFFRL (SEQ ID NO: 4); and MMP2 cleaves AIPVSLR (SEQ ID NO: 46), SLPLGLWAPNFN (SEQ ID NO: 47), HPVGLLAR (SEQ ID NO: 48), GPLGVRGK (SEQ ID NO: 49), and GPLGLWAQ (SEQ ID NO: 50), for example. Other cleavage sites listed in Table 1A or 2A may also be employed. Protease cleavage sites and proteases associated with cancer are well known in the art. Oncomine (www.oncomine.org) is an online cancer gene expression database, so when the agent of the invention is for treating cancer, the skilled person may search the Oncomine database to identify a particular protease cleavage site (or two protease cleavage sites) that will be appropriate for treating a given cancer type. Alternative databases include the European Bioinformatic Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk/gxa). Protease databases include PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org/tools/peptidecutter) and PMAP.Cut DB (cutdb.burnham.org).

In some embodiments, at least one cleavage site may be cleaved through a pH-sensitive cleavage reaction inside the unwanted cell. If the targeted T-cell engaging agent is internalized into the cell, the cleavage reaction may occur inside the cell and may be triggered by a change in pH between the microenvironment outside the unwanted cell and the interior of the cell. Specifically, some cancer types are known to have acidic environments in the interior of the cancer cells. Such an approach may be employed when the interior unwanted cell type has a characteristically different pH from the extracellular microenvironment, such as particularly the glycocalyx. Because pH cleavage can occur in all cells in the lysozymes, selection of a targeting agent when using a pH-sensitive cleavage site may require, when desired, more specificity. For example, when a pH-sensitive cleavage site is used, a targeting agent that binds only or highly preferably to cancer cells may be desired (such as, for example, an antibody binding to mesothelin for treatment of lung cancer).

In certain embodiments, at least one cleavage site may be cleaved by a complement-dependent cleavage reaction. Once targeted T-cell engaging agents bind to the unwanted cell, the patient's complement cascade may be triggered. In such a case, the complement cascade may also be used to cleave the inert binding partner from the first T-cell engaging domain by using a cleavage site sensitive to a complement protease. For example, C1r and C1s and the C3 convertases (C4B, 2a and C3b,Bb) are serine proteases. C3/C5 and C5 are also complement proteases. Mannose-associated binding proteins (MASP), serine proteases also involved in the complement cascade and responsible for cleaving C4 and C2 into C4b2b (a C3 convertase) may also be used. For example, and without limitation, C1s cleaves YLGRSYKV (SEQ ID NO: 73) and MQLGRX (SEQ ID NO: 74). MASP2 is believed to cleave SLGRKIQI (SEQ ID NO: 75). Complement component C2a and complement factor Bb are believed to cleave GLARSNLDE (SEQ ID NO: 76).

In some embodiments, at least one cleavage site may be cleaved by a protease that is colocalized to the unwanted cell by a targeting moiety that is the same or different from the targeting moiety in the targeted T-cell engaging agent. For example, any protease may be simultaneously directed to the microenvironment of the unwanted cells by conjugating the protease to a targeting agent that delivers the protease to that location. The targeting agent may be any targeting agent described herein. The protease may be affixed to the targeting agent through a peptide or chemical linker and may maintain sufficient enzymatic activity when bound to the targeting agent.

In some embodiments, both the first component and second component are mispaired with an inert binding partner. In some embodiments, the protease cleavage site in the first component and the second component are the same. In other embodiments, the protease cleavage sites in the first component and the second component are different cleavage sites for the same protease. In other embodiments, the protease cleavage sites in the first component and the second component are cleavage sites for different proteases. In some embodiments employing two different proteases, the unwanted cell expresses both proteases.

In some embodiments, in a first component, the inert binding partner in an uncleaved state interferes with the specific binding of a VL or VH T-cell engaging domain to its partner VH or VL, respectively, T-cell engaging domain in a second component. In some embodiments, the inert binding partner in an uncleaved state inhibits the binding of the VL or VH T-cell engaging domain to its partner VH or VL, respectively, T-cell engaging domain in a second component such that the dissociation constant (Kd) of the VL or VH T-cell engaging domain to its partner VH or VL, respectively, T-cell engaging domain in a second component in an uncleaved state is at least 100 times greater than the Kd of the VL or VH T-cell engaging domain to its partner VH or VL, respectively, T-cell engaging domain in a second component in a cleaved state.

E. Linkers

In addition to the cleavage site, linkers may optionally be used to attach the separate parts of the targeted T-cell engaging agents together. By linker, we include any chemical moiety that attaches these parts together. In some embodiments, the linkers may be flexible linkers. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long. In some embodiments, the linker is a peptide linker that has a single or repeating GGGGS (SEQ ID NO: 85), GGGS (SEQ ID NO: 86), GS (SEQ ID NO: 87), GSGGS (SEQ ID NO: 88), GGSG (SEQ ID NO: 89), GGSGG (SEQ ID NO: 90), GSGSG (SEQ ID NO: 91), GSGGG (SEQ ID NO: 92), GGGSG (SEQ ID NO: 93), and/or GSSSG (SEQ ID NO: 94) sequence(s).

In some embodiments, the linker is a maleimide (MPA) or SMCC linker.

F. Methods of Making

The targeted T-cell engaging agents as described herein can be made using genetic engineering techniques. Specifically, a nucleic acid may be expressed in a suitable host to produce a targeted T-cell engaging agent. For example, a vector may be prepared comprising a nucleic acid sequence that encodes the targeted T-cell engaging agent including all of its component parts and linkers and that vector may be used to transform an appropriate host cell.

Various regulatory elements may be used in the vector as well, depending on the nature of the host and the manner of introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired.

Chemical linkage techniques, such as using maleimide or SMCC linkers, may also be employed.

In instances where the binding partner is an aptamer, a person of ordinary skill in the art would appreciate how to conjugate an aptamer to a protein, namely the T-cell engaging domain. Aptamers may be conjugated using a thiol linkage or other standard conjugation chemistries. A maleimide, succinimide, or SH group may be affixed to the aptamer to attach it to the T-cell engaging domain.

II. Pharmaceutical Compositions

The targeted T-cell engaging agents may be employed as pharmaceutical compositions. As such, they may be prepared along with a pharmaceutically acceptable carrier. If parenteral administration is desired, for instance, the targeted T-cell engaging agents may be provided in sterile, pyrogen-free water for injection or sterile, pyrogen-free saline. Alternatively, the targeted T-cell engaging agents may be provided in lyophilized form for resuspension with the addition of a sterile liquid carrier.

III. Methods of Treatment

A. Reduction of Unwanted Cells, Targeting of Immune Response, and Treatment of Cancer

The targeted T-cell engaging agents described herein may be used in a method of treating a disease in a patient characterized by the presence of unwanted cells comprising administering a two-component system comprising at least one targeted T-cell engaging agent and a second component to the patient, as each of the components have been described in detail in various embodiments above. Additionally, the agents described herein may also be used in a method of targeting a patient's own immune response to unwanted cells comprising administering a two-component system to the patient.

The amount of the agent administered to the patient may be chosen by the patient's physician so as to provide an effective amount to treat the condition in question. The first component and the second component of the two-component system may be administered in the same formulation or two different formulations within a sufficiently close period of time to be active in the patient.

The patient receiving treatment may be a human. The patient may be a primate or any mammal. Alternatively, the patient may be an animal, such as a domesticated animal (for example, a dog or cat), a laboratory animal (for example, a laboratory rodent, such as a mouse, rat, or rabbit), or an animal important in agriculture (such as horses, cattle, sheep, or goats).

The condition characterized by unwanted cells may include cancer. The cancer may be a solid or non-solid malignancy. The cancer may be a solid tumor wherein the solid tumor is not a lymphoma. The cancer may be any cancer such as breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease and premalignant disease.

The two-component system may be administered alone or in conjunction with other forms of therapy, including surgery, radiation, or traditional chemotherapy.

EXAMPLES Example 1. Preparation of Constructs

Various constructs, both control and experimental, were prepared and used in the examples.

A. Single Chain scFv Bispecific Constructs

A single chain scFv construct was used in this application in order to serve as a positive control. Construct 6245 (SEQ ID NO: 165) was prepared as a bispecific antibody comprising an anti-EPCAM scFv and anti-CD3E scFv. This construct does not comprise any mispairing with an inert binding partner and has both active targeting and T-cell engaging moieties.

B. Precleaved Two-Component System Constructs Using a Targeting scFv

Construct 6246 (SEQ ID NO: 166) and 6247 (SEQ ID NO: 167) are complementary precleaved constructs in a two-component system. By precleaved, the description refers to a construct with a functional targeting moiety and an unpaired T-cell engaging moiety (i.e., one that is not mispaired to an inert binding partner and one that is also not yet paired with its correct partner to form a functional T-cell engaging complex). Both constructs comprise an anti-EPCAM scFv. Construct 6246 comprises an anti-CD3E VH domain, whereas construct 6247 comprises an anti-CD3E VL domain. Neither construct contains an inert binding partner as a mispairing partner.

C. Two-Component System Constructs Using a Targeting scFv and a T-Cell Engaging Domain Mispaired to an Inert Binding Partner, as Well as a Protease Cleavage Site for Releasing the Inert Binding Partner

Construct 6248 (SEQ ID NO: 168) and 6249 (SEQ ID NO: 169) are complementary constructs in a two-component system. Both constructs comprise an anti-EPCAM scFv. Construct 6248 comprises an anti-CD3E VH domain linked through a 25-mer linker having an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VL domain from gantenerumab. Construct 6249 comprises an anti-CD3E VL domain linked through a 25-mer linker having an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VH domain from clone alpha-MUC1-1 antibody.

D. Two-Component System Constructs with a Targeting Moiety, and a T-Cell Engaging Domain Mispaired with an Inert Binding Partner without a Protease Cleavage Site for Releasing the Inert Binding Partner

Constructs 9327 (SEQ ID NO: 170) and 9328 (SEQ ID NO: 171) are two-component system constructs using an scFv targeting domain; however, they do not have a protease cleavage site for releasing the inert binding partner that serves as a mispairing moiety. Both constructs comprise an anti-EpCAM scFv for targeting the constructs to the unwanted cells expressing EpCAM. Construct 9327 comprises an anti-CD3E VH domain linked by a 25-mer linker that does not have a protease cleavage site corresponding to a protease used in the examples to an inert binding partner VL domain from gantenerumab. Construct 9328 comprises an anti-CD3E VL domain linked by a 25-mer linker that does not have a protease cleavage site corresponding to a protease used in the examples to an inert binding partner VH domain from clone alpha-MUC1-1 antibody.

Because these constructs do not have a protease cleavage site corresponding to a protease used in the examples, the inert binding partner will remain attached to the construct, preventing the two would-be complementary components of the two-component system from coming together to create an active anti-CD3E scFv.

E. Constructs Providing Different Targeting Moieties

Constructs 9329 (SEQ ID NO: 172) and 9330 (SEQ ID NO: 173) provide different targeting moieties. These constructs were intended to be used in two-component systems where one construct targets a first antigen on a cancer cell and the second construct targets a second antigen on the same cancer cell. Because the relative size of scFv and VHH targeting moieties are similar, these constructs were intended to “mix-and-match” with the pairable constructs having an anti-CD3E VL domain.

Construct 9329 comprises an anti-glypican-3 VHH sequence. It also comprises an anti-CD3E VH domain attached by a 25-mer linker comprising an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VL domain from gantenerumab.

Construct 9330 comprises an anti-SDC1 scFv from indatuximab as the targeting moiety. It also comprises an anti-CD3E VH domain attached by a 25-mer linker comprising an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VL domain from gantenerumab.

F. VHH/scFv Bispecific Constructs

Construct 9332 (SEQ ID NO: 174) and 9333 (SEQ ID NO: 175) are both VHH/scFv bispecific constructs comprising an anti-EGFR VHH portion and an anti-CD3E scFv portion. These two constructs do not comprise any insert binding domain.

G. Two-Component System Constructs Using a Targeting VHH Domain, a T-Cell Engaging Domain Mispaired to an Inert Binding Partner and Comprising a Protease Cleavage Site for Releasing the Inert Binding Partner

Constructs 9334 (SEQ ID NO: 176) and 9335 (SEQ ID NO: 177) are complementary two-component system constructs using a targeting VHH domain. Both constructs comprise an anti-EGFR VHH domain for targeting to the unwanted cells expressing EGFR. Construct 9334 comprises an anti-CD3E VH domain linked by a 25-mer linker having an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VL domain from gantenerumab. Construct 9335 was prepared comprising an anti-CD3E VH domain linked by a 25-mer linker having an MMP2 cleavage site (AIPVSLR (SEQ ID NO: 46)) to an inert binding partner VH domain from clone alpha-MUC1-1 antibody.

Thus, as a summary, the constructs are as provided in Table 4, with more detail and sequences provided above in Table 1B, with IBD standing for Inert binding partner.

TABLE 4 Construct Summary T-Cell Targeting Engaging IBD? No. Moiety Moiety? Cleavage? Pair with? 6245 anti-EpCAM anti-CD3E no IBD, no no pairing necessary scFv scFv cleavage for TCE activity (positive control) 6246 anti-EPCAM anti-CD3E no IBD, no pairs with at least 6247 scFv VH cleavage 6247 anti-EPCAM anti-CD3E no IBD, no pairs with at least 6246 scFv VL cleavage 6248 anti-EPCAM anti-CD3E MMP2 pairs with at least 6249 scFv VH cleavage site and inert VL 6249 anti-EPCAM anti-CD3E MMP2 pairs with at least 6248 scFv VL cleavage site and inert VH 9327 anti-EpCAM anti-CD3E no cleavage cannot easily pair with scFv VH site and inert 9328 because no VL cleavage site (negative control) 9328 anti-EpCAM anti-CD3E no cleavage cannot easily pair with scFv VL site and inert 9327 because no VH cleavage site (negative control) 9329 anti-Glypican-3 anti-CD3E MMP2 “mix-and-match” with VHH VH cleavage site pairable constructs and inert VL having an anti-CD3E VL domain 9330 anti-SDC1 anti-CD3E MMP2 “mix-and-match” with scFv from VH cleavage site pairable constructs indatuximab and inert VL having an anti-CD3E VL domain 9332 anti-EGFR anti-CD3E no IBD, no no pairing necessary VHH (7D12) scFv cleavage for TCE activity (positive control) 9333 anti-EGFR anti-CD3E no IBD, no no pairing necessary VHH (9D8) scFv cleavage for TCE activity (positive control) 9334 anti-EGFR anti-CD3E MMP2 pairs with at least 9335 VHH VH cleavage site and inert VL 9335 anti-EGFR anti-CD3E MMP2 pairs with at least 9334 VHH VL cleavage site and inert VH

H. Preparation and Storage of all Constructs

Constructs were generated by DNA2.0 (Newark, Calif.) and expressed in HEK293T cells. Single-stranded oligonucleotides were designed to cover a specified sequence with a C-terminal hexahistidine tag to aid with down-stream purification. The oligonucleotides were chemically synthesized, then assembled using a variety of proprietary protocols depending on the sequence characteristics. In some instances, template independent PCR was used. In some instances, smaller sequences were assembled to create larger sequences by use of standard restriction enzyme digestion and ligase-mediated assembly. The assembled oligonucleotides were then cloned into standard E. coli plasmids and the complete double strand sequence verified by automated Sanger sequencing on ABI hardware. Constructs were expressed by transient transfection in HEK293T cells at the 150 ml scale and antibody fragments purified using affinity chromatography.

Before experiments began, constructs were thawed on ice and aliquoted under sterile conditions into low protein-binding tubes. Aliquots were stored at −80° C. until required. Aliquots were thawed on ice immediately prior to use. Aliquots were used for a maximum of five freeze-thaw cycles.

Example 2. Evaluation Construct Manufacturing

A. FIG. 5A: Evaluation of Constructs by SDS PAGE and Coomassie Blue Staining

Aliquots of antibody were thawed on ice, and diluted in 25 mM Tris pH7.4 to a final concentration of 2.0 mg/ml. If the constructs were already more dilute than this, the dilution step was omitted. An appropriate volume of 6× gel sample buffer (0.5 M Tris pH 6.8, 12% (w/v) SDS, 25% (v/v) glycerol, 5 mM EDTA, 200 mM N-ethylmaleimide) was added to each sample, which was then heated to 90° C. for 10 minutes.

10 μg of each construct was run on a 4-20% pre-cast gradient gel. Once run, gels were fixed for 30 minutes in stain buffer (10% (v/v) acetic acid, 50% (v/v) methanol and 40% (v/v) dH₂O) and then stained for 2 hours in Coomassie blue R-250 (0.25% in stain buffer), followed by de-staining for 2 to 3 hours in stain buffer with several changes of buffer as required. Gels were stored in 7% (v/v) acetic acid before documentation.

Results are shown in FIG. 5A. This shows that the proteins have been made and have very high purity, along with the correct molecular mass predicted by their sequence.

B. FIG. 5B

Additional constructs were evaluated in FIG. 5B. The method used for FIG. 5A was used for 5B.

Constructs 6245 (the bispecific construct not requiring pairing), 6248 and 6248 (pairable constructs with an inert binding partner and an MMP2 cleavage site) were produced adequately. Constructs 6246 and 6247 (not containing an inert binding partner) were produced at low yields and are believed to be unstable. It is likely that the VH/VL pairing is important for fragment stability.

Thus, we believe that the constructs mispaired with an inert binding partner are more stable and easier to manufacture.

C. Yield

The yield of the constructs assessed in FIG. 5B was as follows:

TABLE 5 Yields Construct Yield (mg) 6245 13.29 6246 0.57 6247 3.24 6248 17.54 6249 43.52

Example 3. Evaluation of IFNγ Expression in T-Cells Cells Mixed with Tumor Cell Lines and Treated with Various Constructs

Preparations of single constructs and mixed constructs were tested for their IFNγ expression in order to test the ability of the complementary constructs in a two-component system to elicit a T-cell response.

Background of IFNγ Assays Generally:

Expression of cytokine markers in vitro, such as IFNγ expression, is known to have a predictive value for T cell responses and, thus, predicts in vivo results. As described in Ghanekar et al., Clin Diag Lab Immunol j8(3):628-31 (2001), IFNγ expression in CD8+ T cells measured by cytokine flow cytometry (CFC) is a surrogate marker for the response of cytotoxic T lymphocytes. Ghanekar at 628. Prior work showed that there is a strong correlation between the expression of IFNγ by CD8+ T cells and the activity of CTL effector cells. Ghanekar at 630. Prior work shows that the use of data on IFNγ expression allows greater accuracy in assessing CD8+ T-cell responses in a clinical setting. Id. at 631. This demonstrates that the cytokine expression assays herein were known to have predictive value for in vivo and clinical responses. While the methods herein do not follow the exact method steps of Ghanekar because there are multiple ways to assess IFNγ expression, Ghanekar demonstrates that IFNγ expression is a proxy for T-cell activity.

T Cell Line Culture:

Cytotoxic T cells were used in the IFNγ assays and cultured in RPMI-1640 medium containing 4.0 mM L-glutamine, 1% penicillin and streptomycin, 10% heat-inactivated FBS, 1% heat-inactivated human serum (pooled AB serum, TCS Bioscience) and 1,000 U/ml IL-2. Cells were kept at a density of 1-2×10⁶ cells/ml, and were fed by replacement of three quarters of the medium every 48 hours. They were originally generated by adding 10 ug HLA-A*0201-restricted viral peptide NLVPMVATV (SEQ ID NO: 178) to 10 million peripheral blood mononuclear cells (PBMCs) from an HLA-A*0201+ donor. Cells were cultured in RPMI-1640 medium containing 4.0 mM L-glutamine, 1% penicillin and streptomycin, 10% heat-inactivated FBS and 1% heat-inactivated human serum (pooled AB serum, TCS Bioscience) for four days before the media was changed to include 1000 U/ml IL-2. The T cells were predominantly CD8+ T cells with a small amount of CD4+ T cells as well.

Tumor Cell Line Culture:

The following cell lines were used: SW620, MCF-7, SNU398, and U266. Cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution except SNU398 and U266 cells which were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS, 2 mM glutamine and 1% penicillin/streptomycin solution. SW620 cells are derived from a human colon cancer metastasis. MCF-7 cells are derived from a human breast cancer metastatic site (pleural effusion). SNU398 cells are derived from a human anaplastic hepatocellular carcinoma patient in 1990. U266 cells are derived from a human male multiple myeloma patient secreting IgE.

Impact of Constructs on IFNγ Production:

Adherent cell lines were plated in a 96 well plate (100,000 cells per well) for at least 16 hours. Non-adherent cells (100,000 cells per well) were plated on the day of the experiment by centrifuging the culture at 400×g for 5 minutes and resuspending the cells in T cell medium. 20,000 T cells per well in T cell medium were added. Constructs were made up in T cell medium and added to the cultures. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures. The final volume in the culture was 200 μl per well. Cultures were incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The cultures were centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

IFNγ ELISA:

IFNγ levels in tissue culture supernatants were assayed using either an eBioscience Ready-Set-Go ELISA kit (cat. no. 88-7316-88) or a BioLegend Human IFNγ ELISA Max kit (cat. no. 430106) as per the manufacturer's instructions.

A. FIG. 6

IFNγ was evaluated for various single constructs and mixed constructs. The IFNγ production and ELISA assay protocols provided above were used, except as noted. SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/ml) were made up in T cell medium and added to the culture. Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO2 and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

IFNγ was evaluated for various single constructs and mixed constructs. Single constructs were assessed, with construct 6425 (a bispecific scFv for EpCAM and CD3E) was serving as a positive control. Baseline IFNγ was assessed in T-cells with SW620 cancer cells, SW620 cancer cells alone, and T-cells stimulated nonspecifically with phytohaemagglutinin (PHA) to show the capacity of T-cells for IFNγ expression.

In SW620 tumor cells, constructs were used at a final concentration of 1 μg/ml. Cultures were incubated for 4 hours and the supernatants were assayed for IFNγ. Mean±standard deviation of triplicates are provided. was evaluated for various single constructs and mixed constructs. Cultures were incubated for 4 hours and the supernatants were assayed for IFNγ. Mean±standard deviation of triplicates are provided. Constructs were used at a final concentration of 1 μg/ml. Cultures were incubated for 24 hours and the supernatants were assayed for IFNγ. Mean±standard deviation of triplicates are provided.

Construct 6245 serves as a positive control because this construct has both a targeting anti-EpCAM scFv and an anti-CD3E scFv; thus, it is a bispecific construct not requiring pairing for T-cell engaging (TCE) activity.

Constructs 6248 and 6249 (pairs of a two-component system each having an inert binding partner separated from the anti-CD3E T-cell engaging VL or VH, respectively, by a linker with an MMP2 cleavage site) showed more IFNγ expression when paired together than when administered alone. The combination of 6246 and 6247 (pairs of a two-component system without any mispairing to an inert binding partner or protease cleavage site required for them to associate) yield a much lower response than the combination of 6248 and 6249 likely because the 6246 and 6247 are not protected during manufacturing by the inert binding partner, which is believed to stabilize the unpaired anti-CD3E VH and VL domains in each construct, respectively. Thus, we believe that the mispaired constructs having an inert binding partner are more stable and easier to manufacture than precleaved constructs having an unpaired anti-CD3E VH or VL domain.

B. FIG. 7

IFNγ was evaluated for various single constructs and mixed constructs. SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/ml) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs was 1 μg/ml per construct). Controls were PHA-M (final concentration 10 μg/m), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ. Mean±standard deviation of triplicates are provided.

Construct 6245 serves as a positive control because this construct has both a targeting anti-EpCAM scFv and an anti-CD3E scFv; thus, it is a bispecific construct not requiring pairing for T-cell engaging (TCE) activity.

Constructs 6248 and 6249 (pairs of a two-component system each having an inert binding partner separated from the anti-CD3E T-cell engaging VL or VH, respectively, by a linker with an MMP2 cleavage site) showed more IFNγ expression when paired together than when administered alone. The combination of 6246 and 6247 (pairs of a two-component system without any binding domain or protease cleavage site required for them to associate) yield a much lower response than the combination of 6248 and 6249 likely because the 6246 and 6247 are not protected during manufacturing by the inert binding partner, which is believed to stabilize the unpaired anti-CD3E VH and VL domains in each construct, respectively. Thus, we believe that the mispaired constructs having an inert binding partner are more stable and easier to manufacture than constructs with an unpaired anti-CD3E VH or VL domain.

C. FIG. 8

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration ranging from 1 ng/ml to 1 μg/ml) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs ranged from 1 ng/ml to 1 μg/ml per construct). Controls were SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ. Mean±standard deviation of triplicates were shown in FIG. 8.

Construct 6245 served as a positive control and paired constructs 6248 and 6249 were assessed. Both the control construct and the paired two-component system showed IFNγ expression. This demonstrates that the inert VL and VH domains are being cleaved from constructs 6248 and 6249, respectively, and that these two constructs are pairing to create a complete anti-CD3E scFv, which is capable of engaging T cells.

The two-component system (6248 and 6249) has a lower potency than the bispecific 6245 construct, yet is still in an acceptable range and may actually offer dosing advantages in avoiding side effects.

D. FIGS. 9A-B

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration ranging from 1 ng/ml to 1 μg/ml) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs ranged from 10 ng/ml to 10 μg/ml per construct). Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO2 and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −200° C. until analyzed for IFNγ. Mean±standard deviation of triplicates were shown in FIGS. 9A-B.

Both the control construct and the paired two-component system showed IFNγ expression. This demonstrates that the inert VL and VH domains are being cleaved from constructs 6248 and 6249, respectively, and that these two constructs are pairing to create a complete anti-CD3E scFv, which is capable of engaging T cells.

The two-component system (6248 and 6249) has a lower potency than the bispecific 6245 construct, yet is still in an acceptable range and may actually offer dosing advantages in avoiding side effects.

E. FIG. 10

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/ml) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs was 1 μg/ml per construct). Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ. FIG. 10 provides mean±standard deviation of triplicates.

FIG. 10 shows a very low level of IFNγ expression for constructs with only a VH or VL for the anti-CD3E scFv; however, positive bispecific constructs with a full scFv (9332 and 9333) showed higher IFNγ expression levels.

F. Stoichiometric Assessment of Complementary Constructs of a Two-Component System (FIG. 11)

Complementary constructs of a two-component system (6248 and 6249) were added together in varying ratios, as shown in FIG. 11.

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs were pre-mixed in the specified ratios in T cell medium and added to the culture (final concentration of constructs was 1 μg/ml in total). The two components 6248 and 6249, one containing the VH domain of the CD3 activating moiety (6249) and the other containing the VL domain of the CD3 activating moiety (6248), were pre-mixed at ratios 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100. The mixtures of the two components were added to 100,000 unwanted tumor cells and 20,000 T cells. Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

The results in FIG. 11 demonstrate an increasing activation of T cells as the ratio of the two components reaches equilibrium. When there is an excess of either the component containing the VH domain of the CD3 activating moiety (6249) or the other component containing the VL domain of the CD3 activating moiety (6248), the activation of T cells is decreased as the activation is reliant on both the VH and VL of the CD3 activating moiety coming together. Therefore, IFNγ expression levels were much lower when all or nearly all of the constructs provided were of one type or the other. The highest IFNγ expression level corresponds to the scenario where an equal amount of each of the two complementary constructs were provided. This provides further evidence demonstrating that the IFNγ expression is caused by the two halves of the anti-CD3E scFv coming together from the two constructs in the two-component system.

G. Use of MCF-7 Cells (FIG. 12)

FIG. 12 shows experiments conducted in the MCF-7 tumor cell line. MCF-7 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/ml) were made up in T cell medium and added to the culture. Controls were PHA-M (final concentration 10 μg/ml), MCF-7 cells plus T cells with no additions, and MCF-7 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

Similar results were achieved to the other cell lines used. In FIG. 12, positive control constructs 6245, 9332, and 9333 (each having a full anti-CD3E scFv) showed much higher IFNγ expression levels than any of the single components comprising only a VH or VL domain from the anti-CD3E antibody. FIG. 12 also provides baseline IFNγ expression levels for MCF-7 cells alone or T-cells stimulated nonspecifically with PHA.

H. FIGS. 13-14

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/m) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs was 1 μg/ml per construct). Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

In FIG. 13, the data show that the two-component system of 6248 and 6249 functions as expected because these constructs have an inert binding partner that can be cleaved by an MMP2 cleavage site and pairable anti-CD3E variable domains (one VH in 6248 and one VL in 6249). Constructs 9327 and 9328 do not generate a strong IFNγ signal because neither of these constructs has a cleavage site between the inert binding partner and the anti-CD3E variable domain.

Constructs 9327 and 6248 do not show any activity because they both have VH domains for the anti-CD3E antibody and cannot make a functional anti-CD3E scFv; additionally, 9327 does not have a cleavage site. 9327 and 6249 show a very low level of activity because 9327 has no cleavage site and 6249 has a cleavage site, but the two together can make an anti-CD3E scFv if some low level of spontaneous cleavage occurs.

In FIG. 14, the pairing of constructs 9334 and 9335 (providing biparatopic approach to targeting EFGR, with targeting antibody scFvs to different epitopes on EGFR) did not create an IFNγ signal. It is believed that either the epitopes on EGFR were too far apart for the two anti-CD3E variable domains to reach each other or the epitopes were too close and creating steric hindrance for binding on the antibody side. It is, however, very reasonable to test biparatopic combinations. Another antibody for EGFR can be identified and tested for combinations in this approach.

FIG. 14 also shows targeting two different proteins expressed on the same cancer cell. Construct 6248 binds EpCAM and is successfully paired with 9335, which binds EGFR. Construct 6249 also binds EpCAM and is successfully paired with 9334. This establishes that different molecules on a cancer cell may be targeted, providing yet a further layer of specificity for some embodiments of the two-component system described herein. This also provides further evidence that components 9335 and 9334 work in other contexts and further suggests these components were either too close or too far from each other in their combination with each other described above.

The combination of 9334 and 6249 provides useful information in this figure, demonstrating that dual targeting can be achieved because construct 9334 targets EGFR and 9249 targets EpCAM.

The combination of 9334 and 6248 was not expected to have activity because both constructs comprise a VH from the anti-CD3E antibody and neither construct comprises a VL from that antibody.

I. FIG. 15

SNU398 cells were cultured in RPMI1640 containing 10% FBS, 2 mM glutamine and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/ml) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs was 1 μg/ml per construct). Controls were PHA-M (final concentration 10 gig/ml), SNU398 cells plus T cells with no additions, and SNU398 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

FIG. 15 shows that adding a protease inhibitor reduces the IFNγ expression of the two-component system having a protease cleavage site (constructs 6248 and 6249). The protease inhibitor does not impact the 6245 bispecific construct as cleavage and pairing are not required for activity.

J. FIG. 16

SW620 cells were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin solution. Cells were plated in a 96 well plate (100,000 cells per well) on the day prior to the experiment. On the day of the experiment the medium was aspirated and discarded. 20,000 T cells per well in T cell medium were added. Constructs (final concentration of 1 μg/m) were made up in T cell medium and added to the culture. Where mixtures of constructs were used, these were pre-mixed before addition to the cultures (final concentration of constructs was 1 μg/ml per construct). Controls were PHA-M (final concentration 10 μg/ml), SW620 cells plus T cells with no additions, and SW620 cells without T cells or other additions. Each condition was run in triplicate. The final volume in the culture was 200 μl per well. The culture was incubated for 24 hours at 37° C., 5% CO₂ and 100% relative humidity. The culture was centrifuged at 400×g for 5 minutes and the supernatants aspirated and placed in a separate plate. Supernatants were stored at −20° C. until analyzed for IFNγ.

The functional combination of 9335 and 6248 shows that different kinds of antibody fragments may be combined in a first component and second component, respectively. 9335 employs an anti-EGFR VHH as the targeting moiety and 6248 employs an anti-EPCAM scFv as the targeting moiety.

Example 4. In Vivo Targeting of B Cell Lymphoma Using a Two-Component System

A two-component system comprising a first component and a second component are administered to a patient having lymphoma. The first component comprises Rituximab or an anti-CD22 antibody as a targeting moiety, a VH domain from an antibody binding CD3 as a T-cell engaging domain, a VL domain as an inert binding partner, and the ADAM28 cleavage site KPAKFFRL (SEQ ID NO: 1). The second component also comprises Rituximab or an anti-CD22 antibody as a targeting moiety, the complementary VL domain from an antibody binding CD3 as a T-cell engaging domain, VH as an inert binding partner, and the ADAM28 cleavage site KPAKFFRL (SEQ ID NO: 1). The VH domain from an antibody binding CD3 as a T-cell engaging domain of the first component and the VL domain from an antibody binding CD3 as a T-cell engaging domain of the second component are capable of binding to each other when not bound to an inert binding partner and possessing the activity to engage a T-cell.

The patient is infused with the agent, which targets all B cells, healthy and malignant. Upon binding malignant cells, the agent comes into contact with proteases whereby cleavage of the protease recognition domain releases the inert binding partners from both the first and the second T-cell engaging domains.

The malignant B cells that are bound by the now-activated two-component system complex attracts the host immune system for cytolysis by T-cells due to the presence and activity of the complex of the first and second T-cell engaging domains.

Example 5. Specific Embodiments of Two-Component Systems

A two-component system chosen from System A-E is prepared according to Table 3 and administered to a patient having cancer. If an item is described as optional, the row of the table describes both two-component systems having or not having that item.

TABLE 6 Certain Embodiments of the Two-Component System First Component T-Cell Engaging Cleavage Targeting Moiety Moiety Site Inert binding partner Optional Linker(s) & Location(s) A Antibody targeting V_(H) of Any Any VH domain that For example, GGGGS (SEQ ID No: 85). HER2 antibody ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the targeting CD3 cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). Second Component T-Cell Optional Optional Engaging Cleavage Optional Inert binding Targeting Moiety Moiety Site partner Optional Linker(s) & Location(s) Antibody targeting V_(L) of antibody Any Any VH domain that For example, GGGGS (SEQ ID No: 85). HER2 targeting CD3 ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). First Component T-Cell Engaging Cleavage Targeting Moiety Moiety Site Inert binding partner Optional Linker(s) & Location(s) B antibody targeting V_(H) of Any Any VH domain that For example, GGGGS (SEQ ID No: 85). EGFR, such as antibody ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Cetuximab targeting CD4 cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). Second Component T-Cell Optional Optional Engaging Cleavage Optional Inert binding Targeting Moiety Moiety Site partner Optional Linker(s) & Location(s) antibody targeting V_(L) of antibody Any Any VH domain that For example, GGGGS (SEQ ID No: 85). EGFR, such as targeting CD4 ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Cetuximab cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). First Component T-Cell Engaging Cleavage Targeting Moiety Moiety Site Inert binding partner Optional Linker(s) & Location(s) C antibody targeting V_(H) of Any Any VH domain that For example, GGGGS (SEQ ID No: 45). CD20, such as antibody ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Rituximab targeting CD8 cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). Second Component T-Cell Optional Optional Engaging Cleavage Optional Inert binding Targeting Moiety Moiety Site partner Optional Linker(s) & Location(s) antibody targeting V_(L) of antibody Any Any VH domain that For example, GGGGS (SEQ ID No: 85). CD20, such as targeting CD8 ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Rituximab cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). First Component T-Cell Engaging Cleavage Targeting Moiety Moiety Site Inert binding partner Optional Linker(s) & Location(s) D antibody targeting V_(H) of Any Any VH domain that For example, GGGGS (SEQ ID No: 85). CD22, such as antibody ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Inotuzumab targeting cleavage site of the T-cell engaging targeting moiety, between the targeting CD28 domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). Second Component T-Cell Optional Optional Engaging Cleavage Optional Inert binding Targeting Moiety Moiety Site partner Optional Linker(s) & Location(s) antibody targeting V_(L) of antibody Any Any VH domain that For example, GGGGS (SEQ ID No: 85). CD22, such as targeting ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Inotuzumab CD28 cleavage site of the T-cell engaging targeting moiety, between the targeting domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). First Component T-Cell Engaging Cleavage Targeting Moiety Moiety Site Inert binding partner Optional Linker(s) & Location(s) E antibody targeting V_(H) of Any Any VH domain that For example, GGGGS (SEQ ID No: 85). CD33, such as antibody ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Gemtuzumab targeting T cell cleavage site of the T-cell engaging targeting moiety, between the targeting receptor domain without creating moiety and the inactive T-cell engaging (TCR) any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1). Second Component T-Cell Optional Optional Engaging Cleavage Optional Inert binding Targeting Moiety Moiety Site partner Optional Linker(s) & Location(s) antibody targeting V_(L) of antibody Any Any VH domain that For example, GGGGS (SEQ ID No: 85). CD33, such as targeting T cell ADAM28 binds to the VL domain Located between the V_(H) and V_(L) of the Gemtuzumab receptor cleavage site of the T-cell engaging targeting moiety, between the targeting (TCR) domain without creating moiety and the inactive T-cell engaging any binding specificity domain, and/or between the V_(H) and V_(L) of the inactive T-cell engaging domain (See FIG. 1).

Example 6. Embodiments

The following numbered items provide embodiments as described herein, though the embodiments recited here are not limiting.

Item 1. A two-component system for treating a condition characterized by the presence of unwanted cells comprising:

-   -   a. a first component comprising a targeted T-cell engaging agent         comprising:         -   i. a first targeting moiety that is capable of targeting the             unwanted cells;         -   ii. a first T-cell engaging domain capable of T-cell             engaging activity when binding a second T-cell engaging             domain, wherein the second T-cell engaging domain is not             part of the first component;         -   iii. a first inert binding partner for the first T-cell             engaging domain binding to the first T-cell engaging domain             such that the first T-cell engaging domain does not bind to             the second T-cell engaging domain unless the inert binding             partner is removed; and         -   iv. a cleavage site separating the first T-cell engaging             domain and the first inert binding partner, wherein the             cleavage site is:             -   (1) cleaved by an enzyme expressed by the unwanted                 cells;             -   (2) cleaved through a pH-sensitive cleavage reaction                 inside the unwanted cell;             -   (3) cleaved by a complement-dependent cleavage reaction;                 or             -   (4) cleaved by a protease that is colocalized to the                 unwanted cell by a targeting moiety that is the same or                 different from the targeting moiety in the agent,     -   b. a second component comprising a second T-cell engaging domain         capable of T-cell engaging activity when binding the first         T-cell engaging domain,         wherein the first and second T-cell engaging domains are capable         of binding when neither is bound to an inert binding partner.

Item 2. The two-component system of item 1, wherein the second component further comprises a second targeting moiety that is capable of targeting the unwanted cells.

Item 3. The two-component system of any one of items 1-2, wherein the second component further comprises a second inert binding partner for the second T-cell engaging domain binding to the second T-cell engaging domain such that the second T cell engaging domain does not bind to the first T-cell engaging domain unless the inert binding partner is removed and

-   -   a. a cleavage site separating the second T-cell engaging domain         and the second inert binding partner, wherein the cleavage site         is:         -   i. cleaved by an enzyme expressed by the unwanted cells;         -   ii. cleaved through a pH-sensitive cleavage reaction inside             the unwanted cell;         -   iii. cleaved by a complement-dependent cleavage reaction; or         -   iv. cleaved by a protease that is colocalized to the             unwanted cell by a targeting moiety that is the same or             different from the targeting moiety in the agent,             wherein cleavage of the cleavage site causes loss of the             inert binding partner and complementation with the first             T-cell engaging domain of the two-component system.

Item 4. The two-component system of any one of items 1-3, wherein the first and the second targeting moieties are the same.

Item 5. The two-component system of any one of items 1-3, wherein the first and the second targeting moieties are different.

Item 6. The two-component system of any one of items 1-5, wherein the first and second cleavage site are the same.

Item 7. The two-component system of any one of items 1-5, wherein the first and second cleavage site are different.

Item 8. The two-component system of any one of items 1-7, wherein at least one cleavage site is a protease cleavage site.

Item 9. The two-component system of any one of items 1-8, wherein at least one cleavage site is capable of being cleaved outside the unwanted cells.

Item 10. The two-component system of any one of items 1-9, wherein at least one enzyme expressed by the unwanted cells is a protease.

Item 11. The two-component system of any one of items 1-10, wherein at least one inert binding partner specifically binds the T-cell engaging domain.

Item 12. The two-component system of any one of items 1-11, wherein at least one inert binding partner is a VH or VL domain.

Item 13. The two-component system of any one of items 1-12, wherein

-   -   a. when the T-cell engaging domain is a VH domain, the inert         binding partner is a VL domain and     -   b. when the T-cell engaging domain is VL domain, the inert         binding partner is a VH domain.

Item 14. The two-component system of any one of items 1-13, wherein at least one targeting moiety is an antibody or functional fragment thereof.

Item 15. The two-component system of any one of items 1-14, wherein the at least one inert binding partner is capable of dissociation once at least one cleavage site has been cleaved and after dissociation the two T-cell engaging domains are capable of binding to each other and exhibiting T-cell engaging activity.

Item 16. The two-component system of item 1-15, wherein one T-cell engaging domain is a VH domain and the other T-cell engaging domain is a VL domain.

Item 17. A component for use in a two-component system for treating a condition characterized by the presence of unwanted cells comprising a first targeted T-cell engaging agent comprising:

-   -   a. a targeting moiety that is capable of targeting the unwanted         cells;     -   b. a first T-cell engaging domain capable of T-cell engaging         activity when binding a second T-cell engaging domain, wherein         the second T-cell engaging domain is not part of the first         targeted T-cell engaging agent;     -   c. an inert binding partner for the first T-cell engaging domain         binding to the first T-cell engaging domain such that the first         T-cell engaging domain does not bind to the second T-cell         engaging domain unless the inert binding partner is removed; and     -   d. a cleavage site separating the first T-cell engaging domain         and the inert binding partner, wherein the cleavage site is:         -   i. cleaved by an enzyme expressed by the unwanted cells;         -   ii. cleaved through a pH-sensitive cleavage reaction inside             the unwanted cell;         -   iii. cleaved by a complement-dependent cleavage reaction; or         -   iv. cleaved by a protease that is colocalized to the             unwanted cell by a targeting moiety that is the same or             different from the targeting moiety in the agent,             wherein cleavage of the cleavage site causes loss of the             inert binding partner and allows for complementation with             the second T-cell engaging domain that is not part of the             agent.

Item 18. The component for use in a two-component system of item 17, wherein the cleavage site is a protease cleavage site.

Item 19. The component for use in a two-component system of any one of items 17-18, wherein the cleavage site is capable of being cleaved outside the unwanted cells.

Item 20. The component for use in a two-component system of any one of items 17-19, wherein the enzyme expressed by the unwanted cells is a protease.

Item 21. The component for use in a two-component system of any one of items 17-20, wherein at least one inert binding partner specifically binds the T-cell engaging domain.

Item 22. The component for use in a two-component system of any one of items 17-21, wherein the inert binding partner is a VH or VL domain.

Item 23. The component for use in a two-component system of any one of items 17-22, wherein

-   -   a. when the T-cell engaging domain is a VH domain, the inert         binding partner is a VL domain and     -   b. when the T-cell engaging domain is VL domain, the inert         binding partner is a VH domain.

Item 24. The component for use in a two-component system of any one of items 17-23, wherein the targeting moiety is an antibody or functional fragment thereof.

Item 25. A set of nucleic acid molecules encoding the first and second component of the two component system of any one of items 1-16.

Item 26. A nucleic acid molecule encoding the component for use in a two-component system of any one of items 17-24.

Item 27. A method of treating a disease in a patient characterized by the presence of unwanted cells comprising administering the two-component system of any one of items 1-16 to the patient.

Item 28. A method of targeting an immune response of a patient to unwanted cells comprising administering the two-component system of any one of items 1-16 to the patient.

Item 29. The method of any one of items 27-28, wherein the unwanted cells are cancer cells.

Item 30. The method of item 29, wherein the cancer is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/−5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure. 

What is claimed is:
 1. A kit or composition for treating cancer in a patient comprising: a. a first component comprising a targeted T-cell binding agent comprising: i. a first targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer; ii. a first T-cell binding domain capable of T-cell binding activity when binding a second T-cell binding domain, wherein the second T-cell binding domain is not part of the first component, and wherein the first T-cell binding domain is either a VH domain or VL domain; iii. a first inert binding partner for the first T-cell binding domain binding to the first T-cell binding domain such that the first T-cell binding domain does not bind to the second T-cell binding domain unless the inert binding partner is removed, wherein if the first T-cell binding domain is a VH domain, the inert binding partner is a VL domain and if the first T-cell binding domain is a VL domain, the inert binding partner is a VH domain; and iv. a protease cleavage site separating the first T-cell binding domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding domain from the T-cell binding domain in the presence of a protease: (1) expressed by the cancer; (2) colocalized to the cancer by a targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, a second component comprising a second T-cell binding domain capable of T-cell binding activity when binding the first T-cell binding domain, wherein the first and second T-cell binding domains are capable of binding CD3 or the T cell receptor (TCR) when neither is bound to an inert binding partner, and further wherein if the first T-cell binding domain is a VH domain, the second T-cell binding domain is a VL domain and if the first T-cell binding domain is a VL domain, the second T-cell binding domain is a VH domain.
 2. The kit or composition of claim 1, wherein the second component further comprises a second targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer.
 3. The kit or composition of claim 2, wherein the second component further comprises a second inert binding partner for the second T-cell binding domain binding to the second T-cell binding domain such that the second T cell binding domain does not bind to the first T-cell binding domain unless the inert binding partner is removed, wherein if the second T-cell binding domain is a VH domain, the inert binding partner is a VL domain and if the second T-cell binding domain is a VL domain, the inert binding partner is a VH domain and a. a protease cleavage site separating the second T-cell binding domain and the second inert binding partner, wherein the protease cleavage site is (i) cleaved by a protease expressed by the cancer (ii) cleaved by a protease that is colocalized to the cancer by a targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and complementation with the first T-cell binding domain of the kit or composition.
 4. The kit or composition of claim 3, wherein the first and the second targeting moieties are different.
 5. The kit or composition of claim 3, wherein the protease cleavage sites of the first component and second component are different.
 6. The kit or composition of claim 3, wherein the protease cleavage sites of the first component and second component are cleaved by a protease expressed by the cancer.
 7. The kit or composition of claim 3, wherein the protease cleavage sites of the first component and/or second component are cleaved by a protease that is colocalized to the cancer by a targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent.
 8. The kit or composition of claim 3, wherein each inert binding partner is capable of dissociation once at least one protease cleavage site for each inert binding partner has been cleaved and after dissociation the two T-cell binding domains are capable of binding to each other and exhibiting T-cell binding activity.
 9. A component for use in a kit or composition for treating cancer in a patient comprising a first targeted T-cell binding agent comprising: a. a targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer; b. a first T-cell binding domain capable of T-cell binding activity when binding a second T-cell binding domain, wherein the second T-cell binding domain is not part of the first targeted T-cell binding agent, and wherein the first T-cell binding domain is either a VH domain or VL domain and wherein the first T-cell binding domain and the second T-cell binding domain are capable of binding CD3 or TCR; c. an inert binding partner for the first T-cell binding domain binding to the first T-cell binding domain such that the first T-cell binding domain does not bind to the second T-cell binding domain unless the inert binding partner is removed, wherein if the first T-cell binding domain is a VH domain, the inert binding partner is a VL domain and if the first T-cell binding domain is a VL domain, the inert binding partner is a VH domain; and a protease cleavage site separating the first T-cell binding domain and the inert binding partner, wherein the cleavage site is cleaved by a protease that is colocalized to the cancer by a targeting moiety that is an antibody or antigen binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the second T-cell binding domain that is not part of the agent, further wherein if the first T-cell binding domain is a VH domain, the second T-cell binding domain is a VL domain and if the first T-cell binding domain is a VL domain, the second T-cell binding domain is a VH domain.
 10. A set of nucleic acid molecules encoding the first and second components of the kit or composition of claim
 1. 11. A nucleic acid molecule encoding the first targeted T-cell binding agent of claim
 9. 12. A method of treating cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprising administering the composition of claim 1 to the patient.
 13. A method of treating cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprising administering the composition of claim 3 to the patient.
 14. The method of claim 13, wherein the cancer expressing a tumor antigen that binds the first targeting moiety is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.
 15. A method of targeting T cells expressing CD3 or TCR to cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprising administering the composition of claim 3 to the patient.
 16. The kit or composition of claim 1, wherein the first and second T-cell binding domains are capable of forming an scFv when not bound to an inert binding domain. 